673 
G55m      Ghflmberlain 


Southern  Branch 
of  the 

University  of  California 

Lo8  Angeles 


f 


CT3 


*? 


This  book  is  DUE  on  the  last  date  stamped  below. 


MAY  3      1921 
9661  8  I  iiVW 


MAR  7      1930 


m 


ft 


MAY   1       ^^34 

Tf  6  6     1935 


^JUW 


x« 


idlft 


A9«^ 


3in-8,'20 


^Jtr» 


STATE  nOSriAL  SCHOOL, 


METHODS  IN  PLANT  HISTOLOGY 


METHODS 


IN 


PLANT    HISTOLOGY 


BY 

CHARLES  J.  CHAMBERLAIN,  Ph.D. 

Instructor  in  Botany  in  the  University  of  Chicago 


e/3  2. 


CHICAGO 

Wqz  "ClnlversftB  of  Cbfcaoo  press 
1901 

8\3^ 


COPYRIGHT,   I9OI 

BY    THE    UNIVERSITY    OF    CHICAGO 

CHICAGO,  ILLINOIS 


^^ 


y 


STATE  NORMAL  SCHOOL, 

QK 
C35.-VV 

PREFACE. 

This  book  has  grown  out  of  a  course  in  histological  tech- 
nique conducted  by  the  author  at  the  University  of  Chicago. 
The  course  has  also  been  taken  by  non-resident  students  through 
the  Extension  Division  of  the  University.  The  Methods  were 
published  over  a  year  ago  as  a  series  of  articles  in  the  Journal 
of  Applied  Microscopy,  and  have  called  out  numerous  letters  of 
commendation,  criticism,  suggestion,  and  inquiry.  The  work 
has  been  thoroughly  revised  and  enlarged  by  about  one-half.  It 
is  hoped  that  the  criticism  and  suggestion,  and  also  the  expe- 
rience gained  by  contact  with  both  resident  and  non-resident 
students,  have  made  the  directions  so  definite  that  they  may  be 
followed,  not  only  by  those  who  work  in  a  class  under  the 
supervision  of  an  instructor,  but  also  by  those  who  must  work 
in  their  own  homes  without  any  such  assistance. 

More  space  has  been  devoted  to  the  paraffin  method  than  to 
any  other,  because  it  has  proved  to  be  better  adapted  to  the 
needs  of  the  botanist.  The  celloidin  method,  the  glycerine 
method,  and  free-hand  sectioning  are  also  described,  and  their 
advantages  and  disadvantages  are  pointed  out. 

The  first  part  of  the  book  deals  with  the  principles  of  fixing 
and  staining,  and  the  various  other  processes  of  microtechnique, 
while  in  the  later  chapters  these  principles  are  applied  to  spe- 
cific cases.  This  occasions  some  repetition,  but  the  mere  pres- 
entation of  general  principles  will  not  enable  the  beginner  to 
make  good  mounts. 

The  illustrations  and  notes  in  the  later  chapters  are  not 
intended    to    afford    a  study  of   general    morphology,  but   they 


vi  Methods  in  Plant  Histology 

merely  indicate  to  students  with  a  limited  knowledge  of  plant 
structures  the  principal  features  which  the  preparations  should 
show.  The  photomicrographs  were  made  from  the  author's 
preparations  by  Dr.  W.  H.  Knap,  and  figs.  52,  57,  and  59  were 
drawn  by  Miss  Eleanor  Tarrant ;  all  other  figures  of  plant 
structures  were  made  from  the  author's  drawings. 

Corrections  and  suggestions  will  be  heartily  appreciated. 

Charles  J.  Chamberlain. 
Chicago, 
June  I,  igoi. 


CONTENTS. 

PART  I. 

Pagb 

Chapter  I.     Apparatus i 

Chapter  II.     Reagents 7 

Killing  and  Fixing  Agents 7 

Stains 7 

Formulae  for  Alcohols 9 

Miscellaneous  Reagents 10. 

Arrangement  of  the  Outfit 10 

Chapter  III.     Temporary  Mounts 11 

Chapter  IV.     The  General  Method 13 

Killing  and  Fixing 13 

Washing 14 

Hardening  and  Dehydrating 14 

Clearing 16 

The  Transfer  from  Clearing  Agent  to  Paraffin 16 

The  Paraffin  Bath 17 

Imbedding 18 

Cutting 19 

Fixing  Sections  to  the  Slide 20 

Removal  of  Paraffin 21 

Removal  of  Xylol 21 

The  Transfer  to  the  Stain 21 

Clearing 22 

Mounting  in  Balsam 22 

A  Tentative  Schedule  for  Paraffin  Sections 23 

Chapter  V.     Killing  and  Fixing  Agents ,    .     .  25 

The  Alcohols 25 

The  Chromic  Acid  Group 26 

Picric  Acid 30 

Corrosive  Sublimate 30 

Formalin - 31 

General  Hints  on  Fixing 31 

Chapter  VI.     Staining 33 

The  Hsematoxylins 34 

The  Carmines 39 

The  Anilins 41 


viii  Methods  in  Playit  Histology 

Chapter  VII.     General  Remarks  on  Staining 47 

Chapter  VIII.     Practical  Hints  on  Staining 53 

Chapter  IX.     The  Celloidin  Method 55 

Chapter  X.     The  Glycerine  Method 58 

PART  II. 

Chapter  XI.     Thallophytes  —  Alg^e 63 

Cyanophycese  ,.,.... 63 

Chlorophycese 65 

Phseophyceae 73 

Rhodophycese 75 

Chapter  XII.  Thallophytes  —  Fungi 77 

Schizomycetes 77 

Myxomycetes 78 

Phycomycetes 79 

Ascomycetes 80 

^cidiomycetes 84 

Basidiomycetes 87 

Lichens 88 

Chapter  XIII.     Bryophytes  —  Hepatic^e 89 

Chapter  XIV.     Bryophytes  —  Musci 97 

Chapter  XV.     Pteridophytes  —  Filicine^e 103 

Chapter  XVI.     Pteridophytes  —  Equisetine^e 115 

Chapter  XVII.     Pteridophytes  —  LycopodinejE 117 

Chapter  XVIII.     Spermatophytes  —  Gymnosperms 119 

Chapter  XIX.     Spermatophytes  —  Angiosperms 129 

Chapter  XX.     Labeling  and  Cataloguing  Preparations  ...  141 

Chapter  XXI.     A  Class  List  of  Preparations  . 143 

Chapter  XXII.     Formula  for  Reagents 149 

Index  . 157 


Part  I. 
CHAPTER  I. 


The  following  list  of  apparatus  includes  a  fair  equipment  for 
histological  work :  a  microscope  magnifying  at  least  400  diam- 
eters;  a  hand  microtome;  a  sliding  microtome  ;  a  razor;  a  hone 
and  a  good  razor  strop ;  a  paraffin  bath  and  lamp  ;  a  turn-table  ; 
a  scalpel ;  a  pair  of  needles  ;  a  pair  of  scissors  ;  a  pair  of  for- 
ceps;  stender  dishes;  minots  or  watch-glasses  ;  a  wash  bottle ; 
a  graduate  (50  or  100  cc);  pipettes;  slides,  1X3  inches;  round 
covers,  18  mm.  or  ^  inch  in  diameter;  and 
square  covers,  7^  inch.  Long  covers,  22  X 
50  mm,,  will  be  needed  for  some  of  the  serial 
sections. 

A  convenient  and  effective  microscope 
should  have  a  rack  and  pinion  coarse  adjust- 
ment, a  fine  adjustment,  two  eyepieces  (about 
one-inch  and  two-inch  preferred),  a  low-power 
objective  of  two-thirds  of  an  inch  or  a  one-inch 
focus,  a  high-power  objective  of  one-fifth  or 
one-sixth  of  an  inch  focus,  a  double  nose- 
piece,  an  iris  diaphragm,  and  an  Abbe  con- 
denser. A  cheap  and  practical  form  is  shown 
in  fig.  /,  and  similar  instruments  are  for  sale 
by  all  the  leading  companies. 

The  hand  microtome  {fig.  2)  will  be  found 
extremely  useful,  especially  by  the  busy  teacher 
who  has  large  classes.  Any  sliding  microtome,  if  kept  in  good 
order,  will  be  sufficient  for  the  work  to  be  described  in  this  book, 
but  those  of  medium  size  are  to  be  preferred.  The  student's 
microtome  {figs,  j  and  j  A)  is  quite  inexpensive  and  does  good 


Fig.  2.  A  convenient  form 
of  hand  microtome. 


Methods  in  Plant  Histology 


Fig.  I.    A  compound  microscope,  with  rack  and  pinion  coarse  adjustment,  micrometer  screw  fine 
adjustment,  triple  nosepiece,  iris  diaphragm,  and  condenser. 


Apparatus 


work.  Where  ex- 
pense is  not  an 
objection  the  Jung 
Thoma.the  Minot, 
and  other  micro- 
tomes of  similar 
grade  are  to  be 
preferred. 

The  stout  ra- 
zors our  grand- 
fathers used  to 
shave  with  are  ex- 
cellent for  free- 
hand sectioning, 
for  hand-micro- 
tome work,  and 
even    for    cutting 


Fig.  3.     The  student's  microtome. 


Fig.  3  A .     Clamp  to  hold  an  ordinary  razor  in  the  student's  microtome. 


Methods  ifi  Pla?it  Histology 


paraffin  sections  on  the  sliding  microtome.  The  blade  should 
be  flat  on  one  side  [fig.  4  A).  Modern  razors  [fig.  4  B)  with 
delicate  blades,  though  good  to  shave  with,  are  worthless  for 
cutting  sections  of  plants.  The  razor  is  a  necessity;  if  a  micro- 
tome knife  is 
wanted  in  addi- 
tion, it  should 
have  a  bevel 
about  like  that 
shown  in  fig.  4  A. 
F'G-4-  Ashortblade.two 

or  three  inches  in  length,  is  to  be 
preferred  to  the  longer  ones,  which 
are  much  more  troublesome  to 
sharpen. 

There  are  numerous  forms  of 
the  paraffin  bath.  Those  with  a 
water-jacket,  a  thermometer,  and  a 
thermostat  to  maintain  an  even 
temperature  are  the  most  con- 
venient   where    gas     is    available 


Fig.  5.     A  paraffin  bath,  with  water-jacket, 
designed  to  be  used  with  a  thermostat. 


Fig.  6.    ^,  top  view;  >B,  sidev.ew;  C", 
tain  the  paraffin. 


id  view ;  D,  box  to  c 


{fig.  j).  As  a  rule  it 
is  easier  to  keep  the 
temperature  constant 
in  the  larger  baths. 
A  bath  which,  if  care- 
fully watched,  gives 
the  very  best  results 
can  be  made  by  any 
tinner,  and  is  very  in- 
expensive. The  ac- 
companying figures 
show  the  form  and 
dimensions  [fig.  6). 

It  is  made  of  cop- 
per one  thirty-second 


Apparatus 


of  an  inch  thick,  but  thicker  copper  is  as  good  or  better.  There 
should  be  two  boxes  to  contain  the  paraffin  ;  the  covers  to  the 
boxes  should  fit  loosely.     Any  kind  of  a  lamp  may  be  used. 

Stender  dishes  are  now  very  generally  used  for  staining  on 
the  slide.  The  form  shown  \xi  fig.  y  A  is  made  just  large  enough 
to  hold  two  slides,  placed 
back  to  back,  and  .  hence 
requires  only  a  minimum  of 
the  reagent.  The  cap  in 
this  form  does  not  fit  closely 
enough  to  keep  absolute 
alcohol  and  xylol,  but  does 
very  well  for  the  other  alco- 
hols and  stains.  We  do  not 
believe  that  the  convex 
cover  is  as  good  as  a  flat 
one.  The  form  shown  in  fig.  y  B  is 
hoi  and  xylol,  but   even  with  this 


Fig.  7.    A,  Naples  jar;  Z>,  stender  dish. 


the  best  for  absolute  alco- 
it   is   better  to  put  a  little 
vaseline  or  glycerine  on  the  cover  to  prevent  any  evaporation. 

Wide-mouthed 
bottles,  though 
not  so  conveni- 
ent, give  just  as 
good  results. 

A  serviceable 
form  of  turn-table 
for  glycerine 
mounts  is  shown 
in  fig.  8. 

The  other 
pieces  of  appa- 
ratus mentioned 
need  no  com- 
ment. By  consulting  a  catalogue,  which  will  be  furnished  by  any 
dealer,  the  beginner  can  determine  what  he  needs  to  buy,  and  what 
he  can  find  substitutes  for,  if  it  is  necessary  to  be  very  economical. 


CHAPTER  II. 


REAGENTS. 


It  would  require  entirely  too  much  space  even  to  enumerate 
the  reagents  which  are  occasionally  used  in  a  fully  equipped 
university  laboratory.  The  following  list  includes  only  those 
which  are  used  constantly.  The  Microtomist' s  Vade-Mecum  by 
Lee  contains  very  complete  formulae  for  stains  and  other  reagents. 
The  quantities  mentioned  below  indicate  about  what  the  average 
student  uses  in  a  three-months'  course  in  methods.  Nearly  all 
the  stains,  however,  would  last  for  a  year,  if  properly  used. 

KILLING  AND  FIXING  AGENTS. 
Commercial  alcohol  (about  95  per  cent.),  2  liters;  absolute 
alcohol,  300  cc;  ether,  50  cc;  chromic  acid,  10  g.;  corrosive 
sublimate,  10  g.;  glacial  acetic  acid,  25  cc;  hydrochloric  acid, 
50  cc;  picric  acid,  5  g.;  chloroform,  50  cc;  [osmic  acid,  i  per 
cent,  solution  in  water,  25  cc.  This  is  extremely  expensive,  and 
not  necessary  except  for  the  most  delicate  work].  Formulae  for 
making  killing  and  fixing  agents  from  these  materials  will  be 
given  later. 

STAINS. 

Only  a  few  of  the  most  important  stains  are  given  in  this 
list.  In  general  one  should  have  enough  of  a  stain  to  stand 
about  two  inches  high  in  the  stender  dish  or  bottle  in  which  the 
staining  is  to  be  done.  The  theory  and  practice  of  staining  will 
be  discussed  later. 

Delafield's  Haematoxylin. — To  100  cc  of  a  saturated  solution  of 
ammonia  alum  add,  drop  by  drop,  a  solution  of  i  g.  haematoxylin 
dissolved  in  6  cc.  of  absolute  alcohol.  Expose  to  air  and  light 
for  one  week,  then  filter.  Add  25  cc  of  glycerine  and  25  cc  of 
methyl  alcohol.  Allow  to  stand  until  the  color  is  rather  dark. 
Filter,  and  keep  in  a   tightly  stoppered  bottle.     The  solution 

7 


8  Methods  m  Plant  Histology 

should  stand  for  two  months  before  it  is  ready  for  use,  but,  if 
needed  immediately,  the  "  ripening,"  which  is  brought  about  by 
the  oxidation  of  hsematoxylin  into  haematin,  may  be  secured  in 
a  few  minutes  by  a  judicious  addition  of  peroxide  of  hydrogen, 

Mayer's  Haem-Alum. — Dissolve  with  gentle  heat  i  g.  of  haema- 
toxylin  in  50  cc,  of  95  per  cent,  alcohol;  add  a  solution  of  50  g. 
of  alum  in  a  liter  of  distilled  water.  Allow  the  mixture  to  cool 
and  settle ;  filter ;  add  a  crystal  of  thymol  to  preserve  from 
mold.  The  stain  is  ready  for  use  as  soon  as  made,  and  it  keeps 
well. 

Haidenhain's  Iron  Alum-Haematoxylin. — Two  solutions  are 
used,  and  they  are  never  to  be  mixed  : 

(a)  A  I  ^  to  4  per  cent,  aqueous  solution  of  ammonia  sulphate  of  iron. 

{b)  A  ^2  per  cent,  aqueous  solution  of  haematoxylin. 

Cyanin,  Erythrosin,  Safranin,  Gentian  Violet. —  Numerous 
formulae  are  given  for  these  and  other  anilin  stains,  but  the  fol- 
lowing general  formula  gives  excellent  results : 

Make  a  3  per  cent,  solution  of  anilin  oil  in  distilled  water; 
shake  thoroughly  and  frequently  for  a  day;  add  enough  alcohol 
to  make  the  whole  mixture  about  20  per  cent,  alcohol.  Add  i  g. 
of  cyanin  or  erythrosin,  etc.,  as  the  case  may  be,  to  100  cc.  of 
the  solution.  Safranin  is  often  used  in  a  strong  alcoholic  solu- 
tion, and  even  with  the  above  formula  it  is  better  to  dissolve  the 
safranin  in  strong  alcohol  before  adding  it  to  the  mixture. 

Acid  Fuchsin. —  Use  a  i  to  2  per  cent,  solution  in  water,  or 
70  per  cent,  alcohol. 

Iodine  Green. —  Use  a  i  to  4  per  cent,  solution  in  water  or 
alcohol.  A  3  per  cent,  solution  in  70  per  cent,  alcohol  is  very 
good  for  the  vascular  system  of  plants. 

Mixtures  of  Fuchsin  and  Iodine  Green. — The  following  formula 
is  often  used  for  karyokinetic  figures  : 

(a)  A  }i  per  cent,  solution  of  fuchsin  in  water. 

{b)  A  %,  per  cent,  solution  of  iodine  green  in  water. 

Just  before  using  mix  {a)  and  (<^)  in  various  proportions  until 
what  is  needed  for  the  particular  case  is  found. 
Orange  G. —  Use  a  saturated  aqueous  solution. 


Reagetits 


Eosin. —  A  I  to  5  per  cent,  solution  in  water  or  alcohol.  A 
2  per  cent,  aqueous  solution  is  good  for  material  to  be  mounted 
in  glycerine,  but  a  2  per  cent,  solution  in  70  per  cent,  alcohol  is 
better  for  balsam  mounts.  The  stronger  solution  may  be  diluted 
as  needed  for  special  cases. 

FORMULAE  FOR  ALCOHOLS. 

The  grades  of  alcohol  in  most  common  use  are  35  per  cent., 
50  per  cent.,  70  per  cent.,  85  per  cent.,  95  per  cent.,  and  100  per 
cent.  The  100  per  cent,  is  expensive,  and  great  care  should  be 
taken  to  keep  the  bottle  well  corked  or  the  stender  dish  closely 
covered.  The  following  formulae  will  enable  anyone  to  make 
the  other  grades  of  alcohol  from  95  per  cent,  alcohol  and  water: 
95    35  95     50  95     70  95    85 


60  45  25 

The  above  are  the  formulae  for  35  per  cent.,  50  per  cent.,  70 
per  cent.,  and  85  per  cent,  alcohol.  Any  other  grade  can  be 
gotten  in  the  same  way.  In  the  first  formula,  subtract  35  from 
95  ;  the  result,  60,  is  the  number  of  cubic  centimeters  of  water 
which  must  be  added  to  35  cc.  of  95  per  cent,  alcohol 
in  order  to  obtain  35  per  cent,  alcohol.  The  mixture 
contains  95  cc.  of  35  per  cent,  alcohol.  If  more  or 
less  than  95  cc.  of  the  mixture  is  needed,  take 
proportional  parts  of  35  and  60.  This  simple  method 
is  a  time-saver,  but  if  the  bottles  or  stender  dishes 
are  to  be  filled  frequently,  it  will  be  a  still  further 
saving  of  time  to  use  a  long  label  [fig.  9) ,  and,  after 
pouring  in  the  95  per  cent,  alcohol,  draw  a  line 
showing  how  high  it  reaches,  and  then,  after  pouring 
in  the  water,  draw  another  line.  The  next  time  it  is 
necessary  to  fill  the  bottles  merely  pour  in  95  per 
cent,  alcohol  until  it  reaches  the  first  line,  and  then  pour  in  water 
until  it  reaches  the  second  line.  It  is  not  necessary  to  use  dis- 
tilled water,  if  pure  drinking  water  is  available. 


Fig.  9. 


10  .  Methods  in  Plant  Histology 

CLEARING  AGENTS. 

Xylol  is  the  most  generally  useful  clearing  agent  yet  known. 
Clove  oil,  cedar  oil,  bergamot  oil,  carbolic  acid,  and  turpentine 
are  all  necessary  for  special  purposes.  About  200  cc.  of  xylol, 
50  cc.  of  clove  oil,  and  25  cc.  of  each  of  the  others  makes  a  fair 
outfit  to  begin  with. 

MISCELLANEOUS. 

Canada  balsam,  25  cc;  glycerine,  50CC.;  glycerine  jelly,  25  cc; 
2  per  cent,  celloidin,  50  cc;  10  per  cent,  celloidin,  50  cc;  hard 
and  soft  paraffin, 500  g.  each  ;  gold  size,  25  cc;  and  a  small  soft 
brush  for  ringing  glycerine  mounts. 

ARRANGEMENT  OF  THE  OUTFIT  FOR  STAINING  AND  MOUNTING. 

It  is  best  to  keep  the  various  reagents  in  definite  positions  in 
order   that  no  time   may  be  lost  in  hunting  for  anything.     The 

UiJ^Vj  (Uiyr\  U>^^  (h^Wj  U%H«tJ  (^.^nftU  Ur^2\ 

TcWOa.J     (Orony&J   L^\J^  \%<if^^^    /atiUU. J  /f,TjtWuvj  HUA-HootJ 
UsiloU    Moo  J   MS   j  (^85  J  ^70^  (^50  j   Cli\ 


UL^  (%.\j^ 


accompanying  diagram    {jig.  10)  of  a  part  of  the  top  of  a  table 
shows  a  convenient  arrangement. 

The  alcohols  are  in  front  and  the  stains  are  placed  behind. 
The  eosin,  fuchsin,  iodine  green,  cedar  oil,  and  clove  oil  may  be 
kept  in  bottles;  the  rest  should  be  in  stender  dishes. 


CHAPTER  III. 


TEMPORARY  MOUNTS. 


Before  considering  the  complicated  methods  involved  in 
making  permanent  preparations  a  word  should  be  said  about 
temporary  mounts.  A  preliminary  examination  of  almost  any 
botanical  material  may  be  made  without  any  fixing,  imbedding, 
or  staining.  If  a  little  starch  be  scraped  from  a  potato,  and  a 
small  drop  of  water  and  a  cover-glass  be  added,  a  very  good 
view  will  be  obtained,  and  if  a  small  drop  of  iodine  solution  be 
allowed  to  run  under  the  cover,  the  preparation,  while  it  lasts, 
could  hardly  be  improved.  The  unicellular  and  filamentous 
algae  can  be  studied  quite  satisfactorily  from  such  mounts. 
The  protonema  of  mosses  and  the  prothallia  of  ferns  should  be 
studied  in  this  way,  even  if  a  later  study  from  sections  is 
intended.  If  the  top  of  a  moss  capsule  be  cut  off  at  the  level 
of  the  annulus,  a  beautiful  view  of  the  peristome  may  be  obtained 
by  simply  mounting  in  a  drop  of  water,  or,  in  a  case  like  this 
where  no  collapse  is  to  be  anticipated,  the  object  may  be  mounted 
in  a  small  drop  of  glycerine — just  enough  to  come  to  the  edge 
of  the  cover  without  oozing  out  beyond  —  and  the  preparation 
made  permanent  by  sealing  with  gold  size  or  any  good  cement. 
The  antheridia  and  archegonia  of  mosses  may  be  examined  if 
the  surrounding  leaves  are  carefully  teased  away  with  needles. 
Free-hand  sectioning  with  a  sharp  razor  and  judicious  teasing 
with  a  pair  of  needles  will  give  a  fair  insight  into  the  anatomy 
of  the  higher  plants  without  demanding  any  further  knowledge 
of  technique.  This  rough  work  is  a  very  desirable  antecedent  to 
the  study  of  microtome  sections,  because  most  students  see  in  a 
series  of  microtome  sections  only  a  series  of  sections  when,  in 
the  mind's  eye,  they  ought  to  see  the  object  building  itself  up  in 
length,  breadth,  and  thickness  as  they  pass  from  one  section  to 
another. 

II 


CHAPTER  IV. 

THE  GENERAL  METHOD. 

We  shall  now  consider  the  routine  of  mounting  an  object  in 
Canada  balsam.  While  the  outline  refers  more  particularly  to 
the  paraffin  method,  the  principles  are  general  in  their  applica- 
tion and  must  be  mastered  by  everyone  who  desires  to  make 
first-class  preparations.  Several  of  the  topics,  like  killing  and 
fixing,  staining,  etc.,  will  be  treated  in  detail  when  considering 
the  various  reagents. 

I.     KILLING  AND  FIXING. 

Usually  the  same  reagent  is  used  for  both  killing  and 
fixing.  The  purpose  of  a  killing  agent  is  to  bring  the  life- 
processes  to  a  sudden  termination,  while  a  fixing  agent  is  used 
to  fix  the  cells  and  their  contents  in  as  nearly  the  living  condi- 
tion as  possible.  The  fixing  consists  in  so  hardening  the 
material  that  the  various  elements  may  retain  their  natural  con- 
dition during  all  the  processes  which  are  to  follow.  This  step 
is  one  of  extreme  importance.  Take  the  killing  and  fixing 
fluids  into  the  field.  If  one  waits  until  the  material  is  brought 
to  the  laboratory,  there  may  be  some  fixing,  but  it  will, 
in  many  cases,  be  too  late  to  do  much  killing.  Material  like 
Spirogyra,  however,  may  be  brought  from  the  field  into  the 
laboratory  before  fixing,  if  considerable  water  be  brought  with 
it.  Branches  with  developing  buds  may  be  brought  in  and  kept 
in  water.  Always  have  the  material  in  very  small  pieces,  in 
order  that  the  reagents  may  act  quickly  on  all  parts  of  the 
specimens.  Pieces  larger  than  one-fourth  inch  cubes  shpuld  be 
avoided  whenever  possible.  For  very  fine  work  no  part  of  the 
specimen  should  require  the  reagent  to  penetrate  more  than 
one-sixteenth  of  an  inch.  In  general,  the  volume  of  the  reagent 
should   be  ten  to  fifty  times  that  of  the  material.     The   time 

13 


14  Methods  in  Plant  Histology 

required  for  this  process  varies  with  the  reagent,  the  character 
of  the  tissue,  and  the  size  of  the  piece.  About  twenty-four 
hours  is  a  commonly  recommended  period  for  chromic  acid 
solutions.  While  this  might  suffice  in  some  cases,  we  should 
recommend  two  or  three  days,  and  even  a  longer  period  would 
probably  do  no  harm. 

II.     WASHING. 

Nearly  all  fixing  agents,  except  the  alcohols,  must  be 
washed  out  from  the  material  as  completely  as  possible  before 
any  further  steps  are  taken,  because  some  reagents  leave  annoy- 
ing precipitates  which  must  be  removed,  and  others  interfere 
with  subsequent  processes.  Aqueous  fixing  agents  with  chromic 
acid  as  their  principal  ingredient  are  washed  out  with  water ; 
aqueous  solutions  of  corrosive  sublimate  are  also  washed  out 
with  water ;  but  alcoholic  solutions  should  be  washed  out  with 
alcohol  of  about  the  same  strength  as  the  fixing  agent ;  picric 
acid,  or  fixing  agents  with  picric  acid  as  an  ingredient,  must  not 
be  washed  out  with  water,  but  with  alcohol,  whether  the  picric 
acid  be  in  aqueous  or  alcoholic  solution.  Running  water  is  best, 
and  where  this  is  not  convenient  the  water  should  at  least  be 
changed  quite  frequently.  The  washing-out  process  usually 
takes  from  twelve  to  twenty-four  hours,  but  it  can  be  shortened 
about  one-half  by  keeping  the  fluid  lukewarm. 

III.     HARDENING  AND  DEHYDRATING. 

After  the  material  has  been  washed,  it  is  necessary  to  con- 
tinue the  hardening  and  also  to  remove  the  water.  Alcohol  is 
used  almost  entirely  for  these  purposes.  It  completes  the 
hardening  and  at  the  same  time  dehydrates,  that  is,  it  replaces 
the  water  in  the  material,  an  extremely  important  considera- 
tion, for  the  least  trace  of  moisture,  a  trace  so  slight  as  to  be 
almost  imaginary,  is  nevertheless  sufficient  to  make  a  prepara- 
tion poor  or  indifferent  when  it  might  have  been  excellent. 

The  process  of  hardening  and  dehydrating  must  be  gradual. 
If  the  material  should  be  transferred  directly  from  water  to 
absolute    alcohol,    the    hardening    and    dehydrating   would    be 


General  Method  15 

brought  about  in  a  very  short  time,  but  the  violent  osmosis 
would  cause  a  ruinous  contraction  of  the  more  delicate  parts. 
Therefore,  transfer  from  water  to  35  per  cent,  alcohol,  which 
should  act  for  six  to  twenty-four  hours.  Then  use  50  per  cent. 
for;  a  similar  period.  Material  may  now  be  placed  in  70  per 
cent,  alcohol,  where  it  may  remain  until  ready  for  use,  since  70 
per  cent,  alcohol  is  a  good  preservative.  Various  devices,  like 
constant  drips  and  osmotic  apparatus,  have  been  proposed  to 
secure  a  more  gradual  transfer.  Whether  these  have  any  real 
advantages  still  remains  to  be  proved.  The  writer  has  taken 
well-fixed  fern  prothallia  through  the  series  35  per  cent.,  50  per 
cent.,  70  per  cent.,  without  the  slightest  plasmolysis.  Such 
things  as  fern  prothallia,  filamentous  algae,  etc.,  can  be  watched 
under  the  microscope  as  the  transfer  is  made,  and,  if  plasmolysis 
results,  the  series  of  alcohols  may  be  made  closer,  e.g.,  10  per 
cent.,  20  per  cent.,  30  per  cent.,  etc.  It  is  said  that  material 
left  for  some  time  in  70  per  cent,  alcohol  will  shrink  in  spite  of 
good  killing  and  fixing,  and  it  is  also  claimed  that  its  capacity 
for  staining  is  diminished.  Some  recommend  that  glycerine  be 
added  to  the  alcohol;  others  prefer  to  complete  the  dehydrating 
process  and  leave  the  material  in  an  essential  oil ;  while  still 
others  would  imbed  it  and  keep  it  in  paraffin.  The  last  is  doubt- 
less best  of  all,  but  requires  such  an  immense  amount  of  labor 
that  it  is  impracticable  for  general  purposes.  Nearly  all  of  our 
own  material  which  is  not  needed  for  immediate  use  is  in  70 
per  cent,  alcohol,  unless,  of  course,  the  material  has  been  put 
into  formalin  or  some  such  reagent  which  kills,  fixes,  and  pre- 
serves all  at  once. 

After  the  70  per  cent,  alcohol,  use  85  per  cent,  and  95 
per  cent.,  allowing  six  to  twenty-four  hours  for  each.  Then 
use  100  per  cent,  alcohol  for  one  or  two  days.  The  70  per 
cent,  would  probably  complete  all  the  hardening  which  is  neces- 
sary, but  the  other  three  must  be  used  to  complete  the  removal 
of  water. 

Up  to  this  point  the  processes  are  exactly  the  same,  whether 
the  material  is  to  be  imbedded  in  paraffin  or  celloidin. 


1 6  Methods  in  Pla?it  Histology 

IV.     CLEARING. 

Let  us  suppose  that  the  material  has  been  thoroughly 
dehydrated,  so  that  not  the  slightest  trace  of  water  remains.  If 
the  supposition  chances  to  be  contrary  to  fact,  all  the  work 
which  has  preceded,  as  well  as  all  which  is  to  follow,  is  only  an 
idle  waste  of  time.  The  purpose  of  a  clearing  agent  is  to  make 
the  tissues  transparent,  but  clearing  agents  also  replace  the 
alcohol.  At  this  stage  the  latter  process  is  the  essential  one, 
the  clearing  which  accompanies  it  being  incidental.  The  clear- 
ing, however,  is  very  convenient,  since  it  shows  that  the  alcohol 
has  been  replaced  and  that  the  material  is  ready  for  the  next  step. 

Various  clearing  agents  are  in  use.  Xylol  is  the  most  gen- 
erally employed,  and  for  most  purposes  it  seems  to  be  the 
best.  Bergamot  oil,  cedar  oil,  clove  oil,  turpentine,  and  chloro- 
form are  all  necessary  for  special  purposes. 

The  transfer  from  absolute  alcohol  to  the  clearing  agent 
should  he  gradual,  like  the  hardening  and  dehydrating  processes. 
The  following  is  a  good  method : 

3  parts  loo  per  cent,  alcohol  and  i  part  xylol,  i  to  lo  hours. 
2  parts  100  per  cent,  alcohol  and  2  parts  xylol,  i  to  lo  hours. 
I  part    I  GO  per  cent,  alcohol  and  3  parts  xylol,  i  to  10  hours. 

This  transfer  is  best  accomplished  by  adding  the  xylol  to  the 
alcohol.  It  is  not  necessary  to  measure,  since  anyone  can  guess 
with  suflficient  accuracy.  Shake  gently  each  time  the  xylol  is 
added.  Pour  off  the  mixture  and  add  pure  xylol,  which  should 
cause  the  material  to  become  transparent.  This  may  require 
only  a  moment,  but  may  require  hours.  Other  clearing  agents 
may  be  used  in  the  same  way  instead  of  the  xylol. 

V.  THE  TRANSFER  FROM  CLEARING  AGENT  TO  PARAFFIN. 
This  should  also  be  a  gradual  process.  The  most  con- 
venient method  is  to  place  a  small  block  of  parafifin  in  the  pure 
clearing  agent  with  the  material.  The  parafifin  dissolves  gradu- 
ally and  produces  the  same  result  as  if  a  small  shaving  of 
paraffin  had  been  added  every  few  minutes  for  a  day  or  so. 
During  this  process  the  bottle  or  dish  should  be  kept  lukewarm. 
Six  to  ten  hours,  or  over  night,  is  usually  sufficient  for  this  step, 


General  Method  ly 

although  it  would  seem  that  material  may  be  kept  here  for  a 
much  longer  time  without  injury,  and  in  case  of  such  refractory 
material  as  the  megaspores  of  the  heterosporous  pteridophytes 
four  or  five  days  seem  necessary.  Excellent  preparations  of  the 
embryo-sac  of  Aster  have  been  made  from  material  which  had 
remained  in  the  xylol  and  paraffin  for  nearly  three  years.  No 
more  paraffin  should  be  added  than  will  go  into  perfect  solution. 
The  temperature  may  be  gradually  increased,  so  that  a  much 
greater  amount  of  paraffin  will  go  into  solution,  but  the  paraffin 
must  not  be  allowed  to  crystallize. 

VI.     THE  PARAFFIN  BATH. 

This  step  is  usually  called  infiltration,  but  when  the  trans- 
fer from  the  clearing  fluid  to  paraffin  is  made  gradually,  as  has 
just  been  indicated,  the  process  of  infiltration  is  already  begun. 
It  is  now  necessary  to  get  rid  of  the  xylol  or  other  clearing 
agent.  This  may  be  done  by  simply  pouring  off  the  mixture  of 
xylol  and  paraffin  and  replacing  it  with  pure  melted  paraffin. 
The  bath  should  be  kept  at  a  temperature  about  i°  C.  higher 
than  the  melting-point  of  the  paraffin.  Fifty-three  degrees  C. 
is  a  good  temperature  for  general  purposes,  but  this  may  be 
reduced  from  i°  to  3°  C.  in  winter,  and  must  often  be  raised 
in  summer.  For  special  purposes  it  is  sometimes  necessary  to 
use  a  temperature  as  high  as  70°  C.  If  the  xylol  or  other 
clearing  agent  is  not  thoroughly  removed,  the  paraffin  will  be 
granular  or  mealy,  and  will  not  cut  well.  The  paraffin  should  be 
changed  once  or  twice  to  make  sure  that  the  clearing  agent 
is  all  removed.  Do  not  waste  this  paraffin,  for  the  clearing 
agent  can  be  driven  off  by  prolonged  heating,  and  the  paraffin 
will  be  better  than  ever.  Most  people  use  soft  paraffin  (about 
45°  C.)  for  the  first  half  of  the  time  necessary  for  infiltration, 
and  a  harder  grade  (48°  C.  to  54°  C.)  for  the  latter  part  of  the 
process.  This  is  a  good  plan,  for  soft  paraffin  melts  at  a  lower 
temperature,  and  it  is  always  well  to  minimize  heat. 

Some  think  that  it  is  better  not  to  pour  off  the  mixture  of  the 
clearing  agent  and  paraffin,  but  rather  to  evaporate  the  clearing 


1 8  Methods  i7i  Plant  Histology 

agent  by  keeping  the  temperature  just  high  enough  to  prevent 
the  crystallization  of  the  paraffin.  This  method  has  certainly 
given  good  results  with  small,  delicate  objects. 

The  time  required  for  infiltration  varies  with  the  character 
of  the  tissue  and  the  size  of  the  piece.  Few  things  can  be  well 
infiltrated  in  less  than  an  hour.  Lily  ovaries  require  one  to  four 
hours  ;  heads  of  Aster  at  the  fertilization  period,  six  to  twelve 
hours.  Some  claim  that  even  delicate  objects,  like  fern  pro- 
thallia  and  the  filamentous  algae  and  fungi,  are  not  injured  by 
a  bath  of  several  days,  if  care  be  taken  not  to  let  the  tempera- 
ture rise  above  48°  C.  to  50°  C.  No  one  seems  to  know  how 
long  a  certain  object  should  remain  in  the  bath,  but  many 
competent  investigators  are  now  using  more  prolonged  periods. 

VII.     IMBEDDING. 

Material  may  be  imbedded  in  paper  trays,  watch  crystals, 
or  in  apparatus  made  for  the  purpose.  Imbedding  L's  consist- 
ing of  two  L-shaped  pieces  {^fig.  11)  of  brass, 
type  metal,  or  lead  are  very  convenient. 
We  use  a  pair-of  L-shaped  pieces  with  arms 
three  inches  long.  These  furnish  a  box  of 
almost  any  required  size.  A  piece  of  glass 
serves  for  a  bottom.  The  tray,  minot,  or 
whatever  is  used,  should  be  slightly  smeared 
with  glycerine,  to  prevent  sticking.  If  several  objects  are  to  be 
imbedded  in  one  dish,  it  is  best  to  have  the  dish  as  near  the 
temperature  of  melted  paraffin  as  possible  ;  otherwise  the  objects 
may  stick  to  the  bottom,  and  it  will  be  impossible  to  arrange 
them  properly.  Great  care  should  be  taken,  however,  not  to 
have  the  dish  too  hot,  since  too  high  a  temperature  not  only 
injures  the  material,  but  also  prevents  a  thorough  imbedding. 
Pour  the  paraffin  with  the  objects  into  the  imbedding  dish  and 
cool  as  rapidly  as  possible.  If  paraffin  cools  slowly,  it  crystal- 
lizes and  does  not  cut  well.  The  layer  of  paraffin  should  be 
just  thick  enough  to  cover  the  objects,  not  only  as  a  matter  of 
economy,  but  because  a  thick  layer  retards  the  cooling.     Very 


General  Method 


19 


small  objects,  like  the  megaspores  of  Marsilea,  ovules  of  Silphium, 
etc.,  may  simply  be  poured  out  upon  a  cool  piece  of  glass.  In 
this  way  very  thin  cakes  are  made,  which  harden  very  i^apidly. 

VIII.  CUTTING. 
Twenty  minutes  after  an  object  is  imbedded  it  is  ready  for 
cutting.  Trim  the  paraffin  containing  the  object  into  a  conveni- 
ent shape,  and  fasten  it  upon  a  block  of  wood.  Blocks  of  pine 
three-fourths  of  an  inch  long  and  three-eighths  of  an  inch  square 
are  good  for  general  purposes.  Put  paraf^n  on  the  end  of  the 
block  so  as  to  form  a  firm  cap  about  one-eighth  of  an  inch  thick. 
Warm  the  cap  and  the  bottom  of  the  piece  containing  the  object, 
and  press  them  lightly  together ;  then  touch  the  joint  with  a  hot 
needle,  put  the  whole  thing  into  cold  water  for  a  minute,  and  it 
is  ready  for  cutting.  Cutting  can  be  learned  only  by  experience, 
but  a  few  hints  may  not  come  amiss :  (^)  Keep  the  knife  sharp. 
If  expense  is  not  too  serious  an  objection,  it  is  well  to  have  two 
hones,  one  rather  soft,  for  use  when  the  knife  is  dull,  and  the 
other  quite  hard,  for  putting  on  an  even  edge.  Flood  the  stone 
with  water,  and  rub  it  with  the  small  slip  which  accompanies  all 
high-grade  hones  ;  this  not  only  makes  a  lather  which  facilitates 
the  sharpening,  but  it  also  keeps  the  surface  of  the  hone  flat. 
As  soon  as  the  edge  of  the  knife  appears  smooth  and  even 
under  a  magnification  of  thirty  or  forty  diameters,  the  sharpen- 
ing is  completed  with  a  good  strop.  It  is  better  to  sharpen  the 
knife  every  time  you  use  it.  {b)  Keep  the  microtome  well  oiled 
and  clea?i.  {c)  Trim  the  block  so  that  each  section  shall  be  a 
perfect  rectangle. 


B 
Fig.  12. 


20  Methods  in  Plant  Histology 

A  ribbon  of  sections  like  that  shown  in  fig.  12  A  is  much 
better  than  one  like  B  of  the  same  figure,  because  sections  will 
usually  come  off  in  neater  ribbons  if  the  knife  strikes  the  longer 
edge  of  the  rectangle,  so  that  the  sections  are  united  by  their 
longer  sides  rather  than  by  the  shorter.  Crooked  ribbons  are 
caused  by  wedge-shaped  sections,  and  are  always  to  be  avoided, 
because  they  make  it  difficult  to  economize  space,  and  also 
because  they  present  such  a  disorderly  appearance.  The  knife, 
which  should  be  placed  at  a  right  angle  to  the  block  and  not 
obliquely,  should  strike  the  whole  edge  of  the  block  at  once,  and 
should  leave  in  the  same  manner. 

IX.     FIXING  SECTIONS  TO  THE  SLIDE. 

Sections  must  be  firmly  fixed  to  the  slide,  or   they  will  be 
washed  off  during  the  processes  involved   in  staining.     Mayer's 
albumen  fixative  is  excellent  for  this  purpose.      Formula : 
White  of  egg,  50  cc.     (Active  principle.) 
Glycerine,  50  cc.     (To  keep  it  from  drying  up.) 
Salycilate  of  soda,  i  g.     (Antiseptic,  to  keep  out  bacteria,  etc.) 

Shake  well  and  filter.  It  will  keep  from  two  to  six  months. 
The  fixative  may  be  used  alone  or  in  connection  with  the  water 
method.  Put  a  small  drop  of  fixative  on  the  slide,  smear  it 
evenly  over  the  surface,  and  then  wipe  it  off  with  a  clean  finger 
or  piece  of  linen  until  only  a  scarcely  perceptible  film  remains ; 
then  add  several  drops  of  distilled  water  and  float  the  sections 
or  ribbons  on  the  water.  Warm  gently  until  the  paraffin 
becomes  smooth  and  free  from  wrinkles.  Be  careful  not  to 
melt  the  parafifin,  for  the  albumen  of  the  fixative  coagulates 
with  less  heat  than  is  required  to  melt  the  paraffin.  It  is  a  very 
good  plan  to  put  the  slide  on  the  top  of  the  water  bath  for  a 
moment  and  then,  after  the  sections  have  become  smooth, 
remove  the  surplus  water  and  leave  them  on  the  bath  with  a 
couple  of  thicknesses  of  blotting  paper  under  them  for  three  or 
four  hours,  or,  better,  over  night.  If  the  fixative  is  used  alone, 
as  is  often  the  case  when  sections  are  very  thick,  none  of  this 
delay  is  necessary,  since  the  sections  are  merely  laid  upon  the 
fixative  and  pressed  down  gently  with  the  finger. 


Ge7ieral  Method  2i 

X.     REMOVAL  OF  THE  PARAFFIN. 

To  remove  the  paraffin  it  is  very  customary  to  heat  the 
slide  gently  until  the  paraffin  melts,  and  then  place  the  slide  in 
xylol  for  thirty  seconds  or  a  minute.  I  believe,  however,  that 
it  is  far  better  merely  to  warm  the  slide  a  little  (not  warmer  than 
30°  C).  Good  xylol  will  then  remove  the  paraffin  in  two  or 
three  minutes.  Even  if  the  slide  should  not  be  warmed  at  all, 
good  xylol  should  remove  the  paraffin  in  five  minutes. 

XI.  REMOVAL  OF  XYLOL. 
The  xylol  may  be  removed  either  by  absolute  alcohol 
or  by  the  95  per  cent.  The  absolute  alcohol  does  not  seem  to 
be  really  necessary.  For  my  own  work  I  have  two  stender 
dishes  of  xylol  and  two  of  absolute  alcohol.  After  the  xylol 
has  been  used  for  a  time  I  employ  it  only  for  removing  paraffin, 
and  in  the  same  way  use  the  absolute  alcohol  only  for  removing 
the  xylol,  while  the  other  two  dishes  are  used  only  for  dehydrat- 
ing and  clearing.  In  transferring  from  xylol  to  absolute  alcohol, 
or  vice  versa,  it  is  well  to  drain  off  the  superfluous  liquid  b^ 
resting  the  corner  of  the  slide  upon  a  piece  of  blotting  paper. 
These  rather  expensive  reagents  will  last  much  longer  when  this 
precaution  is  taken. 

XII.     TRANSFER  TO  THE  STAIN. 

Stains  are  aqueous  or  alcoholic,  and  alcoholic  stains  are 
of  various  strengths.  If  an  aqueous  stain  be  used,  the  slide 
should  be  passed  successively  through  the  alcohols  95  per  cent., 
85  per  cent.,  70  per  cent.,  50  per  cent.,  and  35  per  cent.,  allow- 
ing each  to  act  for  about  thirty  seconds,  after  which  the  slide  is 
put  into  the  stain.  (In  many  cases  it  is  sufficient  to  put  a  few 
drops  of  the  stain  on  the  slide  with  a  pipette.)  From  the  stain 
the  slide  is  passed  back  through  the  various  grades  of  alcohol, 
allowing  it  to  remain  about  thirty  seconds  in  each  as  before.  If 
the  stain  be  alcoholic  of  about  70  per  cent,  strength,  the  process 
is  somewhat  shorter,  for  the  slide  goes  into  the  stain  from  70 
per  cent,  alcohol,  and  goes  back  into  70  per  cent,  alcohol  from 
the  stain.     The  rule  is  to  transfer  to  the  stain  from  the  alcohol 


22  Methods  in  Plant  Histology 

which  is  nearest  the  strength  of  the  stain.  These  directions  are 
based  upon  a  rather  wide  experience  and  certainly  give  excellent 
results  with  stains  which  do  not  forbid  their  application.  How- 
ever, evidence  has  long  been  accumulating  which  indicates  that 
in  case  of  very  thin  sections  (sections  so  thin  that  no  whole  cells 
are  included)  only  the  absolute  alcohol  is  necessary  and  that 
the  rest  may  be  omitted.  Whether  it  is  better  to  omit  the 
alcohols  from  35  per  cent,  to  95  per  cent,  when  staining  on  the 
slide  is  a  question  still  waiting  for  a  definite  answer,  although 
careful  and  systematic  experimenting  would  soon  settle  it. 
Even  if  it  should  be  shown  that  it  is  as  well  or  better  to  omit 
the  alcohols  from  35  per  cent,  to  95.  per  cent,  in  case  of  thin 
sections,  it  must  not.be  inferred  that  these  alcohols  maybe 
omitted  in  preparing  a  piece  of  epidermis  stripped  from  a  leaf, 
for,  in  this  case,  a  ruinous  plasmolysis  occurs,  and  in  sections 
thick  enough  to  include  whole  cells  the  same  damage  is  done. 
In  the  directions  given  in  this  book  it  is  assumed  that  the  series 
35  per  cent.,  50  per  cent.,  70  per  cent.,  85  per  cent.,  95  per  cent., 
and  100  per  cent,  is  to  be  used,  unless  otherwise  mentioned. 
XIII.     CLEARING. 

After  the  sections  have  been  stained,  passed  back  through 
the  various  grades  of  alcohol,  and  have  been  thoroughly  dehy- 
drated in  absolute  alcohol,  they  are  cleared  or  made  transparent 
by  means  of  xylol  or  some  other  clearing  agent.  The  clearing 
agent  must  be  a  solvent  of  balsam.  From  thirty  seconds  to  five 
minutes  will  be  sufficient  for  clearing  any  kind  of  sections. 
XIV.     MOUNTING  IN  BALSAM. 

After  the  sections  are  cleared,  wipe  the  slide  on  the  side 
which  does  riot  bear  the  sections.  Put  on  a  drop  of  Canada 
balsam  and  add  a  clean,*  thin  cover.  Before  the  cover  is  put 
on,  pass  it  through  the  flame  of  an  alcohol  lamp  to  remove 
moisture,  for  it  would  be  a  pity  indeed   to  injure  a  preparation 

'Slides  and  covers  should  be  treated  with  hydrochloric  acid,  or  equal  parts  of 
bydrochloric  acid  and  water,  for  several  hours.  They  should  then  be  thoroughly  rinsed 
in  water  and  placed  in  95  per  cent,  alcohol.  They  should  be  wiped  with  a  cloth  per- 
fectly free  from  lint. 


Geiieral  Method  23 

at  this  stage  of    the  process.     Add  a  label,  and  the  mount  is 
complete. 

A  TENTATIVE  SCHEDULE  FOR  PARAFFIN    SECTIONS. 

It  will  be  useful  to  give  several  tentative  schedules  for  the 
use  of  beginners.  It  cannot  be  too  strenuously  insisted  that 
these  schedules  are  only  tentative,  their  sole  object  being  to  give  the 
beginner  a  start.  The  following  is  a  tentative  schedule  for  the 
ovary  of  a  lily  at  any  period  before  fertilization.  The  pieces 
should  not  be  more  than  half  an  inch  in  length. 

1.  Chromo-acetic  acid,  2  days. 

2.  Wash  in  water,  i  day. 

3.  Thirty-five  per  cent.,  50  per  cent.,  70  per  cent.,  85  per  cent.,  95  per  cent, 
alcohol,  6  to  24  hours  each,  as  convenient. 

4.  One  hundred  per  cent,  alcohol,  24  hours.  This  should  be  changed  once 
or  twice.     The  volume  should  be  at  least  ten  times  that  of  the  material. 

5.  Transfer  from  absolute  alcohol  to  xylol,  allowing  at  least  2  hours  in  each 
of  the  three  mixtures,  and  2  hours  in  pure  xylol. 

6.  Add  paraffin  to  the  xylol  and  keep  warm  for  12  to  24  hours. 

7.  Melted  paraffin  in  the  bath,  2  to  24  hours,  as  convenient.  The  paraffin 
should  be  changed  once  or  twice. 

8.  Imbed. 

9.  Section;  about  lOyu  is  a  good  thickness, 

10.  Fasten  to  the  slide. 

11.  Dissolve  off  the  paraffin  in  xylol. 

12.  One  hundred  per  cent.,  95  per  cent.,  85  per  cent.,  70  per  cent.,  50  per 
cent,  alcohol,  30  seconds  each.  An  hour  in  each  would  do  no  damage 
if  other  duties  should  interfere, 

13.  Delafield's  hematoxylin,  10  minutes, 

14.  Rinse  in  water,  5  minutes.     A  couple  of  hours  does  no  harm. 

15.  Thirty-five  per  cent.,  50  per  cent.,  and  70  per  cent,  alcohol,  30  seconds 
each.     An  hour  would  do  no  harm, 

16.  Acid  alcohol,  i  second, 

17.  Seventy  per  cent,  alcohol,  i  minute.     An  hour  would  do  no  harm. 

18.  Erythrosin,  30  seconds  to  i  minute. 

19.  Eighty-five  per  cent.,  95  per  cent.,  100  per  cent.,  about  5  seconds  each. 
This  step  must  not  be  too  prolonged,  lest  the  erythrosin  wash  out. 

20.  Xylol,  at  least  i  minute.  The  slide  may  be  left  here  for  an  hour  without 
injury. 

21.  M.)unt  in  balsam. 


24  Methods  in  Plant  Histology 

On  the  whole,  it  is  not  a  good  plan  to  use  protracted  periods 
in  the  processes  from  No.  1 1  to  No.  20  inclusive,  because  there 
is  danger  that  a  prolonged  soaking  may  loosen  some  of  the  more 
delicate  contents  of  the  cells,  even  if  whole  sections  do  not 
come  off  bodily. 


CHAPTER   V. 

KILLING  AND  FIXING  AGENTS. 

In  this  short  account  only  the  reagents  which  are  at  present 
considered  most  valuable  for  botanical  work  will  be  considered. 
Probably  no  process  in  microtechnique  is  in  more  urgent  need 
of  improvement  than  this  first  step  of  killing  and  fixing.  Nearly 
all  of  our  formulae  are  merely  empirical,  for  very  few  botanists 
are  expert  chemists,  and  those  who  have  the  requisite  knowl- 
edge of  chemistry  are  interested  in  physiological  problems 
rather  than  in  microtechnique.  The  principal  ingredients  of  the 
usual  killing  and  fixing  agents  are  :  alcohol,  chloroform,  chromic 
acid,  acetic  acid,  osmic  acid,  formic  acid,  picric  acid,  sulphuric 
acid,  platinum  chloride,  iridium  chloride,  corrosive  sublimate, 
and  formalin.     We  shall  consider  first 

THE  ALCOHOLS. 

a.  Ninety-five  per  cent.  Alcohol.  —  This  is  in  quite  general  use 
for  material  which  is  needed  only  for  rough  work.  It  is  extremely 
convenient,  since  it  kills,  fixes,  and  preserves  at  the  same  time 
and  needs  no  changing  or  washing.  It  really  has  nothing  to 
recommend  it  for  fine  work.  It  causes  protoplasm  to  shrink,  but 
cell  walls  usually  retain  their  position,  so  that  95  per  cent,  alco- 
hol will  do  for  free-hand  sections  of  wood  and  many  herbaceous 
stems  ;  but  even  free-hand  sections  of  tender  stems,  like  young 
geraniums  and  begonias,  will  look  better  if  better  reagents  are 
employed.  Alcohols  weaker  than  95  per  cent,  are  not  to  be 
recommended  as  fixing  agents,  although  70  per  cent,  alcohol,  or 
even  50  per  cent.,  will  preserve  material  for  habit  work. 

b.  Absolute  (100  per  cent.)  Alcohol. — This  is  a  fair  killing  and 
fixing  agent,  but  is  rather  expensive.  It  causes  but  little  shrink- 
ing of  the  protoplasm,  and  is  a  time-saver  if  material  is  to  be 
imbedded   in   paraffin.      With   95    per    cent,   or    with    absolute 

25 


26  Methods  in  Plant  Histology 

alcohol,  objects  are  generally  left  in  the  reagent  until  needed  for 
use,  but  such  material  becomes  very  brittle.  The  addition  of 
glycerine  is  an  improvement,  if  material  is  to  be  kept  long. 
Acetic  acid  has  been  used  with  alcohols  to  counteract  the 
tendency  to  shrink.  One  of  the  most  successful  of  the  alcohol 
combinations  is 

c.  Carnoy's  Fluid. — 

Absolute  alcohol,  6  parts. 

Chloroform,  3  parts. 

Glacial  acetic  acid,  i  part. 
The  penetration  of  the  reagent  is  excellent,  and  only  a  few 
hours  are  needed  for  fixing.  Material  should  be  washed  in 
absolute  alcohol  (perhaps  95  per  cent,  alcohol  would  do  no 
harm)  until  there  is  no  odor  of  acetic  acid.  This  should  not 
require  more  than  one  or  two  hours.  It  is  better  to  imbed  in 
paraffin  at  once,  but  when  this  is  not  convenient  the  material 
may  be  transferred  to  85  per  cent,  alcohol  and  then  to  70  per 
cent.,  where  it  maybe  left  until  needed.  Cyanin  and  erythrosin, 
fuchsin  and  iodine  green,  and  similar  combinations,  give  particu- 
larly brilliant  staining  after  this  reagent. 

THE  CHROMIC-ACID  GROUP. 

Chromic  acid,  or  solutions  with  chromic  acid  as  a  foundation, 
are  the  most  generally  useful  killing  and  fixing  agents  yet  known 
to  the  botanist.  A  i  per  cent,  solution  of  chromic  acid  in  water 
gives  good  results,  but  it  is  better  to  use  the  chromic  in  connec- 
tion with  other  ingredients,  such  as  acetic  acid,  formic  acid, 
osmic  acid,  etc.  The  proportions  of  the  various  ingredients, 
for  the  present  at  least,  must  be  determined  by  experiment. 
With  favorable  objects  like  fern  prothailia,  Spirogyra,  and  other 
things  which  can  be  watched  while  the  fixing  is  taking  place, 
suitable  proportions  are  rather  easily  determined,  because  speci- 
mens, after  being  placed  in  the  reagent,  may  be  examined  at 
frequent  intervals,  and  combinations  which  cause  plasmolysis 
may  be  rejected  and  different  proportions  tried  until  satisfactory 
results  are  secured.  For  example,  fern  prothailia  might  be  placed 
in  the  following  solution  :  chromic  acid,  2  g. ;  acetic  acid,  i  cc; 


Killing  and  Fixi?ig  Agents  27 

and  water,  97  cc.  If  plasmolysis  takes  place,  weaken  the  chromic 
or  strengthen  the  acetic,  since  the  chromic  has  a  tendency  to 
produce  contraction,  and  the  acetic  to  cause  swelling.  Too 
large  a  proportion  of  acetic  acid,  however,  may  cause  distortion, 
and  hence  it  would  be  better  to  weaken  the  chromic.  In  case 
of  fern  prothallia,  3  parts  chromic,  i  part  acetic,  and  396  parts 
water  will  cause  practically  no  plasmolysis,  and  the  fixing  is 
sufficiently  thorough  to  permit  imbedding  in  parafifin.  A  com- 
bination may  be  quite  satisfactory  for  fern  prothallia  and  still 
fail  to  give  good  results  with  Spirogyra,  and  a  combination  which 
succeeds  very  well  with  Spirogyra  may  not  succeed  at  all  with  Vau- 
cheria.  For  very  critical  work  the  most  favorable  proportions 
must  be  determined  for  the  particular  plant  under  investigation. 
When  the  effect  of  the  reagent  cannot  be  observed  directly,  it  is 
well  to  make  a  free-hand  section  and  thus  determine  whether 
plasmolysis  takes  place.  It  is  not  safe  to  judge  the  action  of  a 
fixing  agent  by  the  appearance  of  sections  cut  from  material 
which  has  been  imbedded  in  parafifin,  because  shrinking  of  the 
cell  contents  often  takes  place  during  the  transfer  from  absolute 
alcohol  to  the  clearing  agent  or  during  infiltration  with  paraffin, 
and  sometimes  during  even  later  processes.  When  in  doubt  as 
to  proportions,  we  should  suggest  2  cc.  chromic  acid,  2  cc.  acetic 
acid,  and  296  cc.  water  as  a  good  formula  for  most  purposes. 

The  time  required  for  fixing  undoubtedly  varies  with  different 
plants,  but  twenty-four  hours  may  be  considered  a  minimum 
even  for  the  most  delicate  objects.  It  is  well  known  that  zoolo- 
gists allow  fixing  agents  like  Miiller's  fluid  and  Erlicki's  fluid  to 
act  for  weeks  before  the  material  is  passed  on  to  the  next 
stage,  and  it  may  well  be  questioned  whether  botanists  have 
not  made  a  mistake  in  allowing  the  chromic  solutions  to  act  for 
so  short  a  time.  At  present  most  botanists  recommend  sixteen 
to  twenty-four  hours  for  material  which  is  to  be  imbedded  in 
parafifin,  but  some  recent  experiments  in  my  laboratory  indicate 
that  material  which  has  been  in  the  fixing  fluid  for  two  or  three 
days  is  better  able  to  withstand  the  subsequent  processes.  More 
rapid  penetration,  and  consequently  more  immediate  killing,  can 


28  Methods  in  Plant  Histology 

be  secured  if  the  reagent  is  kept  warm  (30°  to  40°  C).  Since 
chromic  acid  has  a  tendency  to  render  objects  hard  and  brittle, 
it  is  often  better  to  use  some  other  fixing  agent,  if  much  diffi- 
culty is  anticipated  in  the  cutting. 

After  fixing  is  complete,  the  reagent  should  be  washed  out 
with  water.  Running  water  is  desirable,  and  where  this  is  not 
convenient  the  water  must  be  changed  frequently.  Any  material 
should  be  sufficiently  washed  in  six  to  twenty-four  hours,  but 
the  time  may  be  shortened  about  one-half  by  using  lukewarm 
water. 

Some  of  the  formulae  are  as  follows  : 

a.  Strong  Chromo-Acetic  Solution. — 

I  g.  chromic  acid. 

I  cc.  glacial  acetic  acid. 

98  cc.  water. 

This  solution  has  been  used  quite  extensively  in  embryo- 
logical  work  upon  the  higher  plants. 

b.  "Weak  Chromo-Acetic  Solution. —  (Schaffner's  formula): 

0.3  g.  chromic  acid. 
0.7  acetic  acid. 

99  cc.  water. 

This  has  also  been  used  in  embryological  work.  It  causes 
little  or  no  plasmolysis,  but  the  chromic  seems  rather  weak. 
Difficult  material,  like  Aster  heads  and  ripe  Capsella  pods,  cuts 
more  readily  after  this  reagent  than  after  the  stronger  solution. 

c.  Medium  Chromo-Acetic  Solution. — 

0.7  g.  chromic  acid. 

0.5  cc.  glacial  acetic  acid. 

100  cc.  water. 

We  are  now  using  this  solution,  and  it  seems  very  successful 
in  nearly  all  cases. 

d.  Flemming's  Fluid. —  (Weaker  solution.) 

(      I  per  cent,  chromic  acid,  25  cc. 

A.  \     ^  P^^  cent,  acetic  acid,  10  cc. 
/     Water,  55  cc. 

B.  I  per  cent,  osmic  acid,  10  cc. 


Killing  and  Fixing  Agents  29 

Keep  the  mixture  A  made  up,  and  add  B  as  the  reagent  is 
needed  for  use,  since  it  does  not  keep  well.  This  fluid  is  quite 
expensive  on  account  of  the  osmic  acid.  For  cytological  work 
it  gives  as  good  results  as  any  fixing  agent  which  has  yet  been 
thoroughly  tested.  It  is  especially  recommended  for  chromo- 
somes, centrosomes,  achromatic  structures,  and  mitotic  phe- 
nomena in  general.  Material  should  be  in  very  small  pieces 
one-eighth  of  an  inch  square,  or  in  thin  slices  one-eighth  of  an 
inch  or  less  in  thickness,  for  the  fluid  penetrates  poorly.  The 
blackening  due  to  the  osmic  acid  may  be  removed  by  peroxide 
of  hydrogen  just  before  the  slide  is  passed  from  the  alcohol  into 
the  stain.  Flemming's  safranin-gentian  violet-orange  combina- 
tion gives  its  most  brilliant  results  after  this  reagent.  It  seems 
possible  that  the  traditional  superiority  of  this  reagent  has  been 
overestimated. 

e.  Merkel's  Fluid. — 

Equal  volumes  of  a  1.4  per  cent,  solution  of  chromic  acid  and  a  1.4  per 
cent,  solution  of  platinic  chloride. 

This  is  also  an  expensive  reagent.  It  is  recommended  for 
mitotic  phenomena,  but  does  not  seem  to  equal  Flemming's 
solution. 

/.  Hermann's  Fluid. — 

1  per  cent,  platinic  chloride,  15  parts. 
Glacial  acetic  acid,  i  part. 

2  per  cent,  osmic  acid,  4  or  2  parts. 

This  is  the  most  expensive  fixing  agent  yet  discovered,  and 
for  botanical  purposes  it  does  not  seem  to  be  any  better  than  the 
cheaper  chromic  mixtures.  It  is  mentioned  here  with  chromic 
mixtures  because  it  originated  as  a  variation  of  Flehiming's 
fluid,  the  platinic  chloride  being  substituted  for  the  chromic 
acid. 

According  to  Lee,  the  chief  objection  to  all  mixtures  into 
which  chromic  acid  enters  is  that  "it  precipitates  certain  of  the 
liquid  albuminoids  of  the  tissues  in  the  form  of  filaments  or  net- 
work, which  are  often  of  great  regularity  and  simulate  structural 
elements  of  the  tissues."    Nevertheless,  the  mixtures  which  have 


30  Methods  in  Plant  Histology 

just  been  described  are  the  best  which  have  yet  been  thoroughly 
tested.  If  material  killed  in  any  of  the  above  mixtures  is  not 
well  washed,  the  haematoxylins  will  not  stain.  It  is  claimed  that 
the  anilins  will  stain  in  spite  of  poor  washing,  but  it  is  a  question 
whether  such  preparations  are  as  permanent  as  those  from  well- 
washed  material. 

PICRIC  ACID. 

Use  a  saturated  solution  in  water  or  70  per  cent,  alcohol. 
One  gram  of  picric  acid  crystals  will  saturate  about  75  cc.  of 
water  or  alcohol.  This  reagent  penetrates  well  and  does  not 
make  the  material  brittle.  It  is  to  be  recommended  when  diffi- 
culty is  anticipated  in  the  cutting.  If  used  cold,  the  time  varies 
from  one  to  twenty-four  hours,  depending  upon  the  character  of 
the  tissue  and  size  of  the  specimen.  If  used  hot  (85°  C),  five 
or  ten  minutes  will  be  sufficient.  Material  should  be  washed  in 
70  or  50  per  cent,  alcohol.  Water  is  injurious,  and  some  even 
go  so  far  as  to  avoid  aqueous  stains,  unless  the  material  has  been 
thoroughly  washed.  The  washing  should  be  continued  until  the 
material  appears  whitish  and  the  alcohol  no  longer  becomes 
tinged  with  yellow.  Picro-carmine  gives  its  best  results  after 
this  reagent.  Picric  acid  can  be  combined  with  various  other 
fixing  agents,  and  so  we  have  picro-sulphuric  acid,  picro-nitric 
acid,  picro-chromic  acid,  picro-chromic-sulphuric  acid,  and  picro- 

osmic  acid. 

CORROSIVE  SUBLIMATE. 

Use  a  2  to  5  per  cent,  solution  in  water,  or  70  per  cent,  alco- 
hol. The  addition  of  about  i  cc.  of  glacial  acetic  acid  to  lOO  cc. 
of  this  solution  is  certainly  an  improvement.  The  time  required 
is  considerably  shorter  than  for  chromic  solutions.  From  one  to 
ten  hours  will  be  found  to  be  sufficient.  If  used  hot  (85°  C), 
only  five  or  ten  minutes  is  required.  Washing  may  be  done 
with  water,  but  50  per  cent,  alcohol  is  better.  If  a  few  drops  of 
an  iodine  solution  be  added  to  the  alcohol,  the  alcohol  takes  on 
a  brownish  color,  but  soon  clears  up.  If  the  addition  of  iodine 
be  continued,  the  washing  is  complete  when  the  alcohol  no 
longer  clears  up,  but  retains  the  brown  color.     If  the  washing  is 


Killing  and  Fixi?ig  Agents  31 

incomplete,  crystals  of  corrosive  sublimate  will  be  unduly  con- 
spicuous in  preparations  made  from  the  material.  Camphor  may 
be  used  instead  of  iodine  to  hasten  the  washing. 

The  carmines  are  very  brilliant  after  corrosive  sublimate  on 
account  of  the  formation  of  mercuric  carminate,  but  haematoxy- 
lin  and  anilines  also  give  good  results.  It  is  claimed,  however, 
that  achromatic  structures  do  not  stain  well  with  the  safranin-gen- 
tian  violet-orange  combination  after  this  fixing  agent,  but  it 
might  be  worth  while  to  test  other  stains  before  discarding  this 
reagent  for  cytological  work. 

Corrosive  sublimate  material  gets  very  brittle  if  allowed  to 
remain  long  in  alcohol^  and  therefore  it  is  better  to  imbed  it  as 
soon  as  possible. 

FORMALIN. 

Formalin  is  a  comparatively  recent  addition  to  the  list  of 
killing  and  fixing  agents.  It  is  an  excellent  preservative,  often 
preserving  the  blue  and  red  colors  as  well  as  the  structure  of 
objects.  A  2  or  4  per  cent,  solution  in  water  is  good  for  fila- 
mentous algs.  The  material  may  simply  be  put  into  the  reagent 
and  left  until  needed  for  use.  For  class  use  formalin  material 
should  be  washed  in  water  for  several  minutes,  because  the 
fumes  are  irritating  to  the  eyes  and  mucous  membranes.  After 
a  thorough  washing  in  water  any  of  the  usual  stains  may  be 
used. 

GENERAL  HINTS  ON  FIXING. 

It  is  very  desirable  that  the  fixing  agent  should  penetrate 
quickly  to  all  parts  of  the  object.  For  this  reason  material 
should  be  in  small  pieces.  The  best  fixing  agents  do  their  best 
work  near  the  surface  of  the  piece.  Small  objects  like  Azolla 
and  fern  prothallia  may  be  thrown  into  the  fixing  agent  entire ; 
even  larger  objects,  which,  like  the  anthers  of  Lilium,  are  easily 
penetrated,  may  also  be  put  in  without  any  cutting.  Most 
objects  larger  than  quarter  of  an  inch  cubes  should  be  trimmed 
with  a  sharp  knife  or  razor ;  some  knowledge  of  the  structures 
concerned  is  essential  before  one  can  trim  material  with  unvary- 
ing success. 


32  Methods  in  Plant  Histology 

Some  objects,  although  small,  cause  trouble  in  various  ways. 
Many  buds  are  hairy  and  will  not  sink  ;  if  such  things  are  dipped 
quickly  in  strong  alcohol,  they  will  usually  sink.  If  rather  large 
air  bubbles  prevent  the  material  from  sinking,  as  in  case  of  peri- 
chaetical  leaves  of  some  mosses  and  involucral  leaves  of  liver- 
worts, a  little  dissection  or  a  careful  snip  with  the  scissors  will 
obviate  the  difficulty.  If  an  air-pump  is  available,  the  bubbles 
are  easily  removed. 

It  is  often  asked  whether  fixing  agents  really  preserve  the 
actual  structure  of  cell  contents.  It  must  be  admitted  that 
some  things,  notably  the  liquid  albuminoids,  are  much  modified 
in  appearance,  but  the  most  competent  observers  are  now 
inclined  to  believe  that  such  delicate  objects  as  chromosomes, 
centrosomes,  the  achromatic  figure,  and  even  the  structure  of 
protoplasm,  can  be  studied  with  confidence  from  material  which 
has  been  fixed,  imbedded,  and  stained.  Recent  study  of  these 
objects  in  the  living  condition  has  strengthened  this  confidence. 

It  is  certain  that  we  have  not  yet  found  the  ideal  fixing 
agent  for  cell  contents.  Such  an  agent  must  not  be  a  solvent 
of  any  of  the  cell  contents,  must  penetrate  rapidly,  must  pre- 
serve structures  perfectly,  and  must  harden  so  thoroughly  that 
every  detail  shall  remain  unchanged  during  the  subsequent 
processes  of  dehydrating,  clearing,  imbedding,  sectioning,  and 
staining. 


CHAPTER  VI. 

STAINING. 

Staining  is  one  of  the  most  important  and  most  complicated 
processes  of  microtechnique.  The  formulae  are  largely  empirical, 
and  there  is  still  abundant  room  for  experimentation  in  the  use 
of  mordants  and  in  the  effect  of  the  same  stain  or  combination 
after  various  fixing  agents. 

Stains  may  be  classified  in  various  ways ;  e.g.,  there  are  three 
great  groups  of  stains  —  the  Carmines,  the  Haematoxylins,  and 
the  Anilins.  Stains  may  be  classified  as  basic  and  acid,  or  they 
may  be  regarded  as  general  and  specific.  A  general  stain  affects 
all  the  elements,  while  a  specific  stain  affects  only  certain 
elements  or  stains  some  elements  more  deeply  than  others. 
Stains  which  show  a  vigorous  afifinity  for  the  nucleus  have  been 
called  nuclear  stains,  and  those  which  affect  the  cytoplasm  more 
than  the  nucleus  have  been  termed  plasma  stains.  Of  course, 
such  stains  are  specific. 

We  shall  consider  some  of  the  more  important  haematoxylins, 
carmines,  and  anilins,  reserving  general  directions  and  theoreti- 
cal questions  until  the  end  of  this  chapter.  Many  of  the  formulae 
are  taken  from  The  Microtomist's  Vade-Mecum  (Lee),  which  is 
easily  the  most  complete  compendium  of  stains  and  other 
reagents  concerned  in  microtechnique.  It  is  to  be  regretted 
that  botanists  have  no  book  of  this  character,  but  it  must  be 
confessed  that  we  have  not  the  material  for  such  an  extensive 
work.  Other  formulae  are  from  Botanical  Microtechnique  (Zim- 
mermann)  and  from  Stirling's  Histology.  The  directions  for 
using  a  stain  apply  to  stains  made  up  according  to  the  formulae 
which  are  given  here,  and  may  need  modification  if  other  formulae 
are  employed.  It  is  hoped,  however,  that  the  directions  will 
give  the  student  sufficient  insight  into  the  rationale  of  staining 
to  enable  him  to  make  any  necessary  modifications. 

33 


34  Methods  in  Plant  Histology 

THE  HJEMATOXYLINS. 

The  most  important  hematoxylins  are  Delafield's  haema- 
toxylin,  Kleinenberg's  haematoxylin,  Erlich's  haematoxylin, 
Boehmer's  haematoxylin,  Mayer's  haem-alum,  and  Haidenhain's 
iron  alum-haematoxylin. 

All  the  haematoxylins  mentioned  above  contain  alum,  and, 
according  to  Mayer,  who  has  written  the  most  important  work 
on  haematoxylin  stains  ("Ueber  das  Farben  mit  Hamatoxylin," 
Mitth.  a.  d.  Zool.  Station  zu  Neapel,  lo  :  170-186,  1891,  and  "  Ueber 
Hamatoxylin,  Carmin  und  verwandten  Materien,"  Zeit.  f.  wiss. 
Mikr.,  16 1  196-220, 1899).  "The  active  agent  in  them  is  a  com- 
pound of  haematin  with  alumina.  This  salt  is  precipitated  in  the 
tissues,  chiefly  in  the  nuclei,  by  organic  and  inorganic  salts  there 
present  [e.  g.,  by  the  phosphates)  and  perhaps  also  by  other 
organic  bodies  belonging  to  the  tissues."  These  salts  are  fixed 
in  the  tissues  by  the  killing  and  fixing  agent,  and  when  the  stam 
is  applied  a  chemical  combination  results,  Haematoxylins  stain 
well  after  any  of  the  fixing  agents  described  in  the  preceding 
paper,  but  they  are  most  effective  when  used  after  members  of 
the  chromic-acid  series. 

Delafield's  Haematoxylin. — "To  100  cc.  of  a  saturated  solution 
of  ammonia  alum  add,  drop  by  drop,  a  solution  of  i  g.  of  haema- 
toxylin dissolved  in  6  cc.  of  absolute  alcohol.  Expose  to  air 
and  light  for  one  week.  Filter.  Add  25  cc.  of  glycerine  and 
25  cc.  of  meth3''l  alcohol.  Allow  to  stand  until  the  color  is 
sufficiently  dark.  Filter,  and  keep  in  a  tightly  stoppered  bottle." 
(Stirling  and  Lee.)  The  addition  of  the  glycerin  and  methyl 
alcohol  will  precipitate  some  of  the  ammonia  alum  in  the  form 
of  small  crystals.  The  last  filtering  should  take  place  four 
or  five  hours  after  the  addition  of  the  glycerine  and  methyl 
alcohol. 

The  solution  should  stand  for  at  least  two  months  before  it 
is  ready  for  using.  This  "ripening"  is  brought  about  by  the 
oxidation  of  haematoxylin  into  haematin,  a  reaction  which  may 
be  secured  in  a  few  minutes  by  a  judicious  application  of  per- 
oxide of  hydrogen. 


Stai?ting  3  5 

Transfer  to  the  stain  from  35  per  cent,  alcohol  or  from  water. 
The  length  of  time  required  is  exceedingly  variable.  Sometimes 
sections  will  stain  deeply  in  three  minutes,  but  it  is  often  necessary 
to  stain  for  thirty  minutes  or  even  longer.  This  stain  may  be 
diluted  with  several  times  its  own  volume  of  water  ;  when  this  is 
done,  the  time  required  is  correspondingly  long,  but  the  staining 
is  frequently  more  precise.  The  length  of  time  required  will  be 
fairly  uniform  for  all  material  taken  from  the  same  bottle.  This 
fact  indicates  that  the  washing  process,  which  follows  killing  and 
fixing,  is  an  important  factor ;  if  the  washing  has  been  thorough, 
the  material  will  stain  readily;  but  if  the  washing  has  been  insuf- 
ficient, the  material  may  stain  slowly  or  not  at  all.  The  washing 
is  particularly  important  when  the  fixing  agent  contains  an  acid. 
Transfer  from  the  stain  to  water.  Distilled  water  is  neither 
necessary  nor  desirable.  Some  writers  recommend  washing  for 
twenty-four  hours,  but  this  seems  unnecessary;  an  hour  is  usually 
enough,  and  a  few  minutes  is  often  sufficient.  Precipitates  are 
often  formed  when  slides  are  transferred  directly  to  alcohol  from 
this  stain  ;  otherwise,  it  would  be  better  to  transfer  from  the 
stain  to  35  per  cent,  alcohol.  Pass  through  the  alcohols  to  70 
per  cent,  alcohol  and  then  give  the  slide  a  few  dips  (two  seconds 
is  often  sufficient)  in  acid  alcohol  (0.5  cc.  HCl.  to  100  cc.  of 
70  per  cent,  alcohol).  This  extracts  the  stain  more  rapidly  from 
other  parts  than  from  the  nuclei,  and  hence  gives  a  good  nuclear 
stain.  Some  prefer  to  stain  for  a  very  short  time  and  use  no 
acid  alcohol,  but,  as  a  rule,  it  seems  best  to  overstain  and  then 
differentiate  in  this  way,  because  sharper  contrasts  are  obtained. 
Transfer  from  acid  alcohol  to  70  per  cent,  alcohol  and  leave  here 
until  a  rich  purple  color  replaces  the  red  due  to  the  acid.  Since 
small  quantities  of  the  acid  alcohol  are  carried  over  into  the  70 
per  cent,  alcohol,  it  is  well  to  add  a  drop  of  ammonia  now  and 
then  to  neutralize  the  effect  of  the  acid.  Too  much  ammonia  is 
to  be  avoided,  for  it  gives  a  disagreeable  bluish  color  with  poor 
differentiation,  probably  on  account  of  the  precipitation  of 
alumina.  The  slide  may  now  be  passed  through  the  alcohols, 
cleared  in  xylol,  and  mounted  in  balsam  ;  or,  if  a  double  stain  be 


36  Methods  in  Plant  Histology 

preferred,  treat  for  thirty  seconds  to  one  minute  with  eosin, 
erythrosin,  or  some  other  stain  affording  a  good  contrast;  rinse 
in  70  per  cent,  alcohol  and  proceed  as  usual. 

Delafield's  hsematoxylin  is  the  most  generally  useful  stain  in 
the  haematoxylin  group.  It  brings  out  cellulose  walls  very 
sharply,  and  consequently  is  a  good  stain  for  embryos  and  the 
fundamental  tissue  system  in  general.  With  safranin  it  forms  a 
good  combination  for  the  vascular  system,  the  safranin  giving 
the  lignified  elements  a  bright  red  color,  while  the  haematoxylin 
stains  the  cellulose  a  rich  purple.  It  is  a  good  stain  for  chro- 
matin, and  the  achromatic  structures  show  up  fairly  well,  but  can 
be  brought  out  much  better  by  special  methods.  Archesporial 
cells  and  sporogenous  tissue  are  very  well  defined  if  proper  care 
be  taken.  Whenever  you  are  in  doubt  as  to  the  selection  of  a 
stain  for  general  purposes,  we  should  advise  the  use  of  Delafield's 
haematoxylin. 

The  following  is  a  general  schedule  for  staining  paraffin  sec- 
tions on  the  slide  in  Delafield's  haematoxylin  : 

1.  Stain  (from  water  or  35  per  cent,  alcohol)  5  minutes. 

2.  Rinse  in  water,  5  minutes. 

3.  Thirty-five  per  cent.,  50  per  cent.,  and  70  per  cent,  alcohol,  30  seconds 
each. 

4.  Acid  alcohol,  i  second. 

5.  Seventy  per  cent,  alcohol,  i  minute. 

6.  Eighty-five  per  cent.,  95  per  cent.,  and  too  per  cent,  alcohol,  30  seconds 
each.  ^ 

7.  Xylol,  I  minute. 

8.  Mount  in  balsam. 

If,  after  rinsing  in  water,  the  stain  is  evidently  too  weak,  put 
the  slide  or  section  back  into  the  stain  until  it  appears  over- 
stained.  For  thin  sections  (lO/^  or  less)  thirty  seconds  in  each 
of  the  alcohols  will  be  unnecessarily  long,  although  thirty  minutes 
would  do  no  damage.  Give  the  slide  a  single  dip  into  the  acid 
alcohol,  transfer  it  quickly  to  the  70  per  cent,  alcohol,  and  then 
examine  it ;  if  it  still  appears  overstained,  give  it  another  dip  in 
the  acid  alcohol,  and  repeat  the  process  until  the  stain  is  what 
you  want.     After  the  haematoxylin  is  just  right,  apply  a  contrast 


Staining  37 

stain,  if  you  wish  to  double-stain.  It  is  a  good  plan  to  move  the 
slide  gently  to  and  fro  in  the  absolute  alcohol.  Before  trans- 
ferring to  the  xylol,  wipe  the  alcohol  from  the  back  of  the  slide, 
or  at  least  rest  the  corner  of  the  slide  upon  blotting  paper 
for  two  or  three  seconds,  in  order  that  you  may  not  carry  over 
so  much  alcohol  into  the  xylol,  and  thus  impair  this  rather 
expensive  reagent.  The  slide  may  also  be  moved  gently  to  and 
fro  in  the  xylol.  Add  a  drop  of  balsam  and  a  cover.  Since  the 
xylol  is  very  volatile,  this  last  step  must  be  taken  quickly.  If 
blackish  spots  appear,  they  are  usually  caused  by  the  drying  of 
sections  before  the  balsam  and  cover  are  added  ;  if  there  are 
whitish  spots  or  an  emulsion-like  appearance,  the  clearing  is  not 
thorough  ;  this  may  be  caused  by  poor  xylol  (or  other  clearing 
agent);  by  absolute  alcohol  which  is  considerably  weaker  than 
its  name  implies  (the  absolute  alcohol  must  test  at  least  as  high 
as  99  per  cent.,  and  ought  to  test  as  high  as  99.5  per  cent.,  if 
xylol  is  to  be  used  for  clearing) ;  or  the  difficulty  may  be  caused 
by  passing  too  quickly  through  the  absolute  alcohol  and  xylol, 
or  may  even  be  caused  by  moisture  on  the  cover-glass. 

Kleinenberg's  Haematoxylin. —  This  stain  has  had  a  wide  use, 
but  it  is  now  largely  replaced  by  better  formulae.  It  is  men- 
tioned here  merely  because  it  is  on  the  shelves  of  so  many 
laboratories!  It  is  doubtful  whether  it  is  equal  to  Delafield's  in 
any  kind  of  botanical  work.  A  good  description  is  given  in  the 
Quarterly  Journal  of  the  Microscopical  Society,  74  :  208  — ,  1897. 
Erlich's  Haematoxylin. — 

Distilled  water,  50  cc. 

Absolute  alcohol,  50  cc. 

Glycerine,  50  cc. 

Glacial  acetic  acid,  5  cc. 

Haematoxylin,  i  g. 

Alum  in  excess. 

Keep  it  in  a  dark  place  until  the  color  becomes  a  deep  red. 
If  well  stoppered,  it  will  keep  indefinitely.  Transfer  to  the  stain 
from  50  per  cent,  or  35  per  cent,  alcohol.  Stain  five  to  thirty 
minutes.     Since  there  is  no   danger  from   precipitates  and  the 


1 


38  Methods  in  Pla?it  Histology 

solution   does  not  overstain,   it  is   not   necessary  to   treat  with 
water  or  with  acid  alcohol,  but  the  slide  may  be  transferred  from 
the  stain  to  70  per  cent,  alcohol.     Eosin,  erythrosin,  or  orange 
G  are  good  contrast  stains. 
Boehmer's  Haematoxylin. — 

Hsematoxylin,  I  g. 

Absolute  alcohol,  1 2  cc. 

Alum,  I  g. 

Distilled  water,  240  cc. 
The   solution  A  must  ripen  for  two  months.     When  wanted 
for  use,  add  about  10  drops  of  A  to  10  cc.  of  B.     Stain  ten  to 
twenty  minutes.     Wash  in  water  and  proceed  as  usual. 

Mayer's  Haem-Alum. —  Haematoxylin,  i  g.,  dissolved  with 
heat  in  50  cc.  of  95  per  cent,  alcohol  and  added  to  a  solution 
of  50  g.  of  alum  in  a  liter  of  distilled  water.  Allow  the  mixture 
to  cool  and  settle ;  filter  ;  add  a  crystal  of  thymol  to  preserve 
from  mold.      (Lee.) 

It  is  ready  for  use  as  soon  as  made  up.  Unless  attacked  by 
mold,  it  keeps  indefinitely.  Transfer  to  the  stain  from  water. 
It  is  seldom  necessary  to  stain  for  more  than  ten  minutes,  and 
four  or  five  minutes  is  generally  long  enough.  As  a  rule,  better 
results  are  secured  by  diluting  the  stain  (about  i  cc.  to  10  cc.  of 
distilled  water)  and  allowing  it  to  act  for  ten  hours  or  over  night. 
This  is  a  good  stain  for  the  nuclei  of  filamentous  algae  and  fungi, 
since  it  has  little  or  no  effect  upon  cell  walls  or  plastids.  Wash 
thoroughly  in  water  and  transfer  to  10  per  cent,  glycerine. 
Specimens  may  be  mounted  in  balsam,  if  they  can  be  gotten 
through  without  shrinking. 

Haidenhain's  Iron  Alum-Haematoxylin. — This  stain  was  intro- 
duced by  Haidenhain  in  1892  and  has  gained  a  well-deserved 
popularity  with  those  engaged  in  cytological  work.  Two  solu- 
tions are  used,  and  they  are  never  mixed : 

A.  One  and  a  half  to  4  per  cent,  aqueous  .solution  of  ammonia  sulphate 
of  iron.     (At  present  we  use  a  3  per  cent,  solution.) 

B.  One-half  per  cent,  aqueous  solution  of  haematoxylin. 

The  first  solution  acts  as  a  mordant,  i.  e.,  it  does  not  stain, 
but  prepares  the  tissue  for  the  action  of  the  second  solution. 


Staining  39 

Transfer  to  the  iron-alum  from  water ;  allow  this  solution  to 
act  for  two  hours ;  wash  in  water  five  minutes,  and  then  stain 
in  the  yi  per  cent,  haematoxylin  ten  hours  or  over  night. 
Rinse  in  water  five  minutes  and  treat  for  a  second  time  with  the 
iron  alum,  which  now  rapidly  extracts  the  stain.  The  action  of 
the  iron  alum  should  be  watched  under  a  microscope,  and  when 
the  chromosomes  of  karyokinetic  figures  appear  sharply  defined, 
the  slide  should  at  once  be  placed  in  water  and  washed  for  at 
least  an  hour,  since  a  very  little  of  the  iron  alum,  if  left  in  the 
tissue,  will  cause  the  preparation  to  fade.  If  staining  for  details 
other  than  nuclei,  the  slide  must  be  transferred  to  water  as  soon 
as  the  desired  effect  is  produced.  After  the  washing  in  water, 
the  slide  is  passed  through  the  alcohols,  cleared  in  xylol,  and 
mounted  in  balsam.  This  stain  is  excellent  for  the  filamentous 
algae  and  fungi,  and  it  keeps  well  in  glycerine.  For  preparations 
to  be  mounted  in  balsam,  erythrosin,  fuchsin,  or  orange  G  make 
good  contrast  stains.  Apply  the  second  stain  after  the  last 
washing  in  water.     The  second  stain  should  always  be  very  light. 

Chromosomes  and  centrosomes  ("of  those  plants  which  have 
centrosomes")  take  a  brilliant  black,  and  other  details,  though 
not  brightly  colored,  often  show  excellent  definition. 

The  times  given  above  must  not  be  accepted  as  final.  Many 
prefer  to  wash  in  water  for  several  hours  after  the  first  immersion 
in  iron  alum.  A  plan  which  has  proved  convenient  and  very 
successful  is  to  put  the  slide  into  the  iron  alum  in  the  morning, 
let  it  wash  in  water  during  the  afternoon,  leave  it  in  the  ^  per 
cent,  of  haematoxylin  over  night,  and  finish  the  preparation  the 
next  morning.  It  is  a  long  process,  requiring  care,  patience,  and 
judgment,  but  it  is  worth  the  effort. 

THE  CARMINES. 

This  group  of  stains,  immensely  popular  a  few  years  ago,  has 
rapidly  lost  favor  among  botanists  as  newer  stains  and  combina- 
tions have  appeared.  When  it  is  desirable  to  stain  in  bulk, 
nothing  has  been  found  which  will  serve  better  than  the  car- 
mines.    Only  three  of  these  stains  will  be  considered  : 


40  Methods  i?i  Plant  Histology 

Greenacher's  Borax  Carmine. — 

Carmine,  3  g. 
Borax,  4  g. 

Distilled  water,  100  cc. 
k 

Dissolve   the   borax   in  water   and  add  the  carmine,  which  is 

quickly  dissolved  with  the  aid  of  gentle  heat.  Add  lOO  cc.  of 
70  per  cent,  alcohol  and  filter.     (Stirling.) 

The  following  is  a  slightly  different  method  for  making  this 
stain  from  the  ingredients  mentioned  above :  Dissolve  the 
borax  in  water,  add  the  carmine,  and  heat  gently  for  ten  minutes  ; 
after  the  solution  cools,  add  the  alcohol  and  filter ;  let  the  solu- 
tion stand   for  two  or  three  weeks,  then  decant,  and  filter  again. 

Stain  the  material  in  bulk  from  50  per  cent,  alcohol  one  to 
three  days,  then  treat  with  acid  alcohol  (50  cc.  of  70  per  cent, 
alcohol  +  2  drops  of  hydrochloric  acid)  until  the  color  becomes 
a  clear  red ;  this  may  require  only  a  few  hours,  but  may  take 
two  or  three  days.  The  material  may  then  be  passed  through 
the  rest  of  the  alcohols  (six  to  twenty-four  hours  each),  cleared, 
imbedded,  and  cut.  After  the  sections  are  thoroughly  fastened 
to  the  slide,  the  paraflfin  should  be  dissolved  off  with  xylol.  The 
balsam  and  cover  may  be  added  immediately,  or  the  slide  may' 
be  passed  down  through  the  alcohols  for  a  contrast  stain. 

Alum  Carmine. — A  4  per  cent,  aqueous  solution  of  ammonia 
alum  is  boiled  twenty  minutes  with  i  per  cent,  of  powdered 
carmine.     Filter  after  it  cools.    (Lee.) 

Stain  from  water  twelve  to  twenty-four  hours  and  wash  in 
water.  No  acid  alcohol  is  needed,  since  the  solution  does  not 
overstain. 

Alum  Cochineal. — 

Powdered  cochineal,  50  g. 

Alum,  5  g. 

Distilled  water,  500  cc. 

Dissolve  the  alum  in  water,  add  the  cochineal,  and  boil ; 
evaporate  down  to  two-thirds  of  the  original  volume,  and  filter. 
Add  a  few  drops  of  carbolic  acid  to  prevent  mold.  (Stir- 
ling.) 


Staining  4 1 

Stain  as  with  alum  carmine.  A  few  years  ago  it  was  a  very 
common  practice  to  stain  in  bulk  in  alum  cochineal  and  counter- 
stain  on  the  slide  with  bismark  brown. 

THE  ANILINS. 

Many  of  the  most  brilliant  and  beautiful  stains  yet  discovered 
belong  to  this  group.  These  stains  are  so  numerous  that  we 
shall  not  attempt  to  mention  even  their  names,  but  shall  con- 
sider only  those  which  are  in  most  common  use  by  botanists. 
The  following  formula  has  proved  to  be  fairly  satisfactory  for  all 
the  anilins  mentioned  in  this  account,  but  other  formulae  will  be 
given  for  most  of  the  stains  : 

Make  a  3  per  cent,  solution  of  anilin  oil  in  distilled  water; 
shake  well  and  frequently  for  a  day;  add  enough  alcohol  to  make 
the  whole  mixture  about  20  per  cent,  alcohol ;  add  i  g.  of  cyanin, 
erythrosin,  safranin,  gentian  violet,  etc.,  to  each  lOO  cc.  of  this 
solution. 

The  anilins  keep  well  in  balsam,  but  not  in  glycerine.  Xylol 
is  a  good  clearing  agent  for  all  of  them,  but  clove  oil  is  very  much 
better  in  case  of  gentian  violet.  Unfortunately,  some  of  them 
do  not  give  permanent  stains.  Some  are  acid,  some  basic,  and 
some  neutral. 

The  rapidity  with  which  sections  must  be  transferred  from 
one  fluid  to  another  makes  many  of  them  more  difficult  to  man- 
age than  the  haematoxylins  or  the  carmines,  but  the  stains  are  so 
valuable  that  even  the  beginner  should  spend  most  of  his  time 
with  the  anilins. 

Many  anilins  stain  quite  deeply  in  one  to  twenty  minutes, 
but  if  the  stain  washes  out  during  the  dehydrating  process,  stain 
longer,  even  ten  to  thirty  hours,  if  necessary.  If  the  stains  are 
made  up  according  to  the  formula  mentioned  above,  transfer  to 
the  stain  from  35  per  cent,  alcohol  and  from  the  stain  to  35  per 
cent,  alcohol,  if  the  stain  does  not  wash  out  too  rapidly;  if  the 
stain  washes  out,  try  50  per  cent.,  70  per  cent.,  85  per  cent.,  95 
per  cent.,  or  even  directly  to  absolute  alcohol.  It  will  often  be 
found  impracticable  to  transfer  from  the  stain  to  alcohols  weaker 


42  Methods  ifi  Plant  Histology 

than  the  85  per  cent.,  lest  all  the  stain  be  washed  out  before  the 
clearing  agent  is  reached. 

Since  the  anilins  are  seldom  used  as  single  stains,  but  almost 
invariably  in  combination  with  other  stains,  the  logical  order 
will  be  disregarded  and  the  stains  will  be  treated,  as  they  are 
used,  in  their  most  usual  combinations. 

Cyanin  and  Erythrosin. —  Stain  in  cyanin  five  to  ten  minutes 
or  longer  ;  rinse  quickly  in  alcohol,  and  then  stain  thirty  seconds 
to  one  minute  in  erythrosin.  If  the  cyanin  washes  out,  stain  for 
an  hour,  and  if  it  still  washes  out,  omit  the  rinsing  in  alcohol 
and  transfer  directly  from  the  cyanin  to  the  erythrosin. 

The  erythrosin  may  be  used  first;  in  this  case  stain  for  five 
minutes  in  erythrosin,  transfer  directly  to  cyanin,  and  stain  for 
about  ten  seconds.  Try  70  per  cent,  alcohol,  but  if  the  stains 
are  lost,  try  85  per  cent.,  95  per  cent.,  or  even  transfer  directly 
to  100  per  cent. 

This  is  a  good  combination  for  general  work,  and  if  properly 
used  is  excellent  for  mitotic  phenomena  and  the  most  delicate 
cytological  work.  Delafield's  haematoxylin  may  be  used  with 
erythrosin.  Stain  first  with  the  haematoxylin,  and,  after  the  pur- 
ple color  has  replaced  the  red  due  to  the  acid,  stain  lightly  with 
erythrosin.  Eosin  may  be  used  instead  of  erythrosin,  but  is  less 
transparent.  Eosin  will  be  mentioned  later  in  connection  with 
special  methods  for  algae  and  fungi. 

Flemming's  Safranin-Gentian  Violet-Orange.  —  Safranin  has 
long  been  a  famous  stain  for  karyokinesis.  This  triple  combina- 
tion was  published  in  1891,  but  its  value  in  plant  cytology  was 
not  thoroughly  appreciated  until  five  or  six  years  later,  when  its 
application  was  developed  to  a  high  degree  of  perfection  by 
various  investigators  of  the  Bonn  (Germany)  school. 

According  to  Flemming,  stain  two  to  three  days  in  safranin 
(dissolve  0.5  g.  safranin  in  50  cc.  absolute  alcohol,  and  after 
four  days  add  10  cc.  distilled  water);  rinse  quickly  in  water ; 
stain  one  to  three  hours  in  a  2  per  cent,  aqueous  solution  of 
gentian  violet ;  wash  quickly  in  water,  and  then  stain  one  to 
three   minutes   in  a  i  per  cent,  aqueous  solution  of  orange   G. 


Staining  43 

Tranfer  from  the  stain  to  absolute  alcohol,  clear  in  clove  oil,  and 
mount  in  balsam. 

The  following  method  seems  to  be  better  for  mitotic  phe- 
nomena in  plants :  Transfer  to  safranin  from  35  per  cent,  alcohol 
and  stain  sixteen  to  twenty-four  hours.  If  the  stain  acts  for 
only  a  few  hours,  it  washes  out  too  rapidly  to  be  controlled  with 
any  precision.  (The  safranin  may  be  made  up  according  to  the 
formula  given  in  the  preceding  paragraph  or  according  to  the 
general  formula.)  Rinse  in  water  for  a  minute  and  then  in  50 
per  cent,  alcohol  until  only  nucleoli  and  the  chromosomes  of 
dividing  nuclei  retain  the  red  color.  If  the  alcohol  does  not 
wash  out  the  stain  sufficiently,  dip  the  slide  in  acid  alcohol  (not 
more  than  2  or  3  drops  of  hydrochloric  acid  to  100  cc.  of 
50  per  cent,  alcohol).  If  acid  has  been  used,  wash  for  a 
moment  in  pure  50  per  cent,  alcohol,  and  then  stain  for  four  or 
five  minutes  in  gentian  violet  (aqueous  solution,  or  made  up 
according  to  the  general  formula).  Rinse  for  a  few  seconds  in 
water  and  then  stain  about  thirty  seconds  in  a  i  per  cent,  aque- 
ous solution  of  orange  G.  Transfer  from  the  stain  directly  to 
absolute  alcohol  and  hasten  the  dehydrating  by  gently  rinsing 
the  slide  in  the  fluid.  One  or  two  quick  dips  in  the  95  per  cent, 
alcohol  before  transferring  to  the  absolute  does  not  seem  to  do 
any  harm  and  certainly  saves  the  more  expensive  reagent.  As  a 
rule,  not  more  than  ten  seconds  can  be  allowed  for  dehydrating, 
because  the  gentian  violet  washes  out  so  rapidly.  The  extreme 
transfer  from  the  orange  G,  an  aqueous  stain,  to  absolute  alco- 
hol, which  must  often  be  made  to  prevent  the  gentian  violet 
from  washing  out,  indicates  that,  for  staining  very  thin  sections 
on  the  slide,  the  series  of  alcohols  from  35  per  cent,  up  to  100 
per  cent,  is  not  at  all  necessary. 

Treat  with  clove  oil  for  five  to  ten  seconds.  The  clove  oil 
not  only  clears,  but  it  rapidly  extracts  the  gentian  violet,  pro- 
ducing an  elegant  differentiation.  Replace  the  clove  oil  by 
cedar  oil,  if  you  have  strictly  first-class  cedar  oil.  Such  cedar 
oil  is  very  thin,  light  yellow  in  color,  and  has  a  faint  odor  of 
cedar  wood.     It  costs  about  the  same  as  clove  oil.     Do  not  get 


44  Methods  hi  Pla?it  Histology 

the  expensive  oil  used  for  immersion  lenses.  If  your  cedar  oil 
is  colorless  and  has  a  strong  odor,  do  not  use  it  at  all,  but  drain 
off  the  clove  oil  as  thoroughly  as  possible  by  resting  the  edge  of 
the  slide  on  blotting  paper  for  two  or  three  minutes,  and  then 
mount  in  balsam.  If  any  considerable  amount  of  clove  oil  is 
left  on  the  slide,  the  preparation  is  almost  sure  to  fade.  The 
cedar  oil  is  not  a  solvent  of  gentian  violet,  and  consequently 
there  is  little  danger  that  the  preparation  will  fade  when  this  oil 
is  used.  Some  investigators  transfer  from  clove  oil  to  xylol, 
but  it  seems  to  us  that  the  brilliancy  of  the  gentian  violet  is 
impaired.  Chromosomes  should  take  a  clear  red  and  the  spindle 
fibers  a  bright  violet.  This  combination  is  generally  used  after 
Flemming's  solution,  but  seems  to  do  equally  well  after  other 
members  of  the  chromic-acid  series.  Achromatic  structures  do 
not  seem  to  stain  well  after  corrosive  sublimate  or  picric  acid. 

Fuchsin. —  Use  a  i  or  2  per  cent,  solution  in  water  or  in  70  per 
cent,  alcohol.  Transfer  to  the  alcoholic  solution  from  70  per 
cent,  alcohol  ;  stain  one  to  two  hours;  differentiate  the  stain  in 
I  per  cent,  solution  of  picric  acid  in  70  per  cent,  alcohol  —  this 
may  require  thirty  seconds  or  several  minutes ;  rinse  in  70  per 
cent,  alcohol  until  a  bright  red  replaces  the  yellowish  color  due 
to  the  acid,  and  then  proceed  as  usual. 

Iodine  Green. —  A  i  per  cent,  solution  in  70  per  cent,  alcohol 
is  good  for  the  vascular  system  of  plants.  This  stain  resists  the 
washing-out  process  better  than  methyl  green.  Stain  in  iodine 
green  at  least  an  hour,  and  it  is  not  a  bad  plan  to  stain  over 
night ;  rinse  in  70  per  cent,  alcohol ;  stain  fifteen  seconds  to  one 
minute  in  erythrosin,  and  proceed  as  usual.  Methyl  green  may 
be  made  and  used  in  the  same  way. 

Fuchsin  and  Iodine  Green  Mixtures. —  Two  solutions  are  kept 
separate,  since  they  do  not  retain  their  efficiency  long  after  they 

are  mixed  : 

.    (  0.1  g.  fuchsin  (acid). 
I  50  cc.  distilled  water. 
„  j  0.1  g.  iodine  green. 
50  cc.  distilled  water. 


Staining  45 

iioo  cc.  absolute  alcohol. 
I  cc.  glacial  acetic  acid. 
0.1  g.  iodine. 
Mix   equal   parts  of  A  and  B.     Transfer  to   the   stain  from 
water.     The   proper  time   must  be  determined   by  experiment. 
Twenty-four  hours  might  be  recommended  for  a  trial.     Transfer 
from  the  stain  directly  to  solution  C  and  from  C  to  xylol. 
Another  formula : 

A.  0.5  g.  acid  fuchsin. 

B.  0.5  g.  iodine  green. 

Mix  a  pipette  full  of  A  with  a  pipette  full  of  B  ;  stain  two  to 
eight  minutes  ;  transfer  to  85  per  cent,  or  95  per  cent,  alcohol, 
dehydrate  rapidly,  clear  in  xylol,  and  mount  in  balsam.  Both 
these  formulae  are  good  for  karyokinesis. 

Bismark  Brown. —  Use  a  2  per  cent,  solution  in  70  per  cent, 
alcohol.  If  material  has  been  stained  in  bulk  in  one  of  the 
carmines,  a  few  minutes'  staining  on  the  slide  with  bismark  brown 
gives  a  good  contrast.      It  is  particularly  good  for  cell  walls. 

Nigrosin. —  Use  a  i  or  2  per  cent,  solution  in  water.  A  few 
drops  of  this  solution  to  a  watch-glass  full  of  water  stains  fila- 
mentous algae  or  fungi  in  one  to  three  hours.  The  stain  keeps 
well  in  glycerine  or  balsam,  but  it  is  hard  to  get  these  forms 
into  balsam  without  more  or  less  shrinking. 


CHAPTER  VII. 

GENERAL  REMARKS  ON  STAINING. 

It  must  be  remembered  that  many  things  may  be  examined 
alive  without  killing,  fixing,  staining,  or  any  of  those  processes. 
A  filament  of  Spirogyra  shows  the  chromatophore  nicely  if 
merely  mounted  in  a  drop  of  water;  the  nucleus  may  be  visible, 
and  the  pyrenoids  can  usually  be  located.  Of  course,  such  a 
study  is  necessary  if  one  is  to  understand  anything  about  the 
plant,  and  in  an  elementary  class  this  might  be  sufficient,  but  a 
drop  of  iodine  solution  applied  to  the  edge  of  the  cover  would 
emphasize  certain  details,  e.g.,  the  starch  in  the  pyrenoids  would 
appear  blue,  the  nucleus  a  light  brown,  and  the  cytoplasm  a 
lighter  brown.  This  illustrates  at  least  one  advantage  to  be 
gained  by  staining  ;  it  enables  us  to  see  structures  which  would 
otherwise  be  invisible,  or  almost  invisible. 

With  so  many  stains  at  our  disposal,  it  at  once  becomes  a 
problem  just  which  stain  or  combination  to  use  in  each  particu- 
lar case.  Beautiful  and  instructive  preparations  occasionally 
result  from  some  happy  chance,  but  uniform  success  demands 
skill  and  judgment  in  manipulation,  and  also  a  knowledge  of  the 
structures  which  are  to  be  differentiated.  Let  us  take  a  vascular 
bundle  for  illustration.  Safranin  stains  the  xylem  a  bright  red, 
but,  with  judicious  washing,  is  entirely  removed  from  the  cam- 
bium and  cellulose  elements  of  the  phloem.  A  careful  staining 
with  Delafield's  haematoxylin  now  gives  a  rich  purple  color  to 
the  cellulose  elements  which  were  left  unstained  by  the  safranin, 
thus  contrasting  sharply  with  the  lignified  elements.  If  cyanin 
and  erythrosin  be  used,  the  xylem  takes  the  blue  and  the  cam- 
bium and  phloem  take  the  red.  Many  terms  have  been  given 
to  indicate  the  affinity  of  certain  tissues  for  certain  stains. 
Auerbach  used  the  terms  "erythrophilous"  and  "cyanophilous" 
in  1890.     This  eminent  zoologist  studied  spermatozoa  and  ova. 

47 


48  Methods  in  Plant  Histology 

He  found  that,  if  preparations  containing  both  spermatozoa  and 
ova  were  stained  with  cyanin  and  erythrosin,  the  nuclei  of  the 
spermatozoa  took  the  cyanin,  while  the  nuclei  of  the  ova  pre- 
ferred the  erythrosin;  hence  the  terms  "cyanophilous "  and 
"erythrophilous."  Auerbach  regarded  these  differences  as  an 
indication  of  sexual  differences  in  the  cells. 

Rosen  (1892)  supported  this  theory,  and  even  went  so  far 
a$  to  regard  the,tube  nucleus  of  the  pollen  grain  as  female,  on 
account  of  its  erythrophilous  staining.  In  connection  with  this 
theory  it  was  suggested  that  the  ordinary  vegetative  nuclei  are 
hermaphrodite,  and  that  in  the  formation  of  a  female  germ 
nucleus  the  male  elements  are  extruded,  leaving  only  the  ery- 
throphilous female  elements  ;  and,  similarly,  in  the  formation  of  a 
male  nucleus  the  female  elements  are  extruded,  leaving  only  the 
cyanophilous  male  elements. 

As  long  ago  as  1884  Strasburger  discovered  that  with  a 
mixture  of  fuchsin  and  iodine  green  the  generative  nucleus  of  a 
pollen  grain  stains  green,  while  the  tube  nucleus  stains  red.  In 
1892  {Verhalten  des  Pollens)  he  discussed  quite  thoroughly  the 
staining  reactions  of  the  nuclei.  The  nuclei  of  the  small  pro- 
thallial  cells  of  gymnosperm  microspores  are  cyanophilous  like 
the  male  generative  nuclei.  The  nuclei  of  a  nucellus  surround- 
ing an  embryo-sac  are  also  cyanophilous,  while  the  nuclei  of 
structures  within  the  sac  are  erythrophilous.  His  conclusion  is 
that  the  cyanophilous  condition  in  both  cases  is  due  to  poor 
nutrition,  while  the  erythrophilous  condition  is  due  to  abundant 
nutrition.  A  further  fact  in  support  of  the  theory  is  that  the 
nuclei  of  the  adventitious  embryos  which  come  from  the  nucellus 
of  Funkia  ovata  are  decidedly  erythrophilous,  while  the  nuclei 
of  the  nucellus  to  which  they  owe  their  food-supply  are  cyano- 
philous. 

In  division  stages  nuclei  are  cyanophilous,  but  from  anaphase 
to  resting  stage  cytoplasm  is  taken  into  the  nucleus,  and  the 
cyanophilous  condition  gradually  changes  to  the  erythrophilous. 

An  additional  fact  in  favor  of  this  theory  is  that  in  Ephedra 
the   tube   nucleus   which   has  very  little  cytoplasm  about   it  is 


General  Remarks  o?i  Stainifig  49 

cyanophilous.  Strasburger  claims  that  there  is  no  essential  dif- 
ference between  male  and  female  generative  nuclei,  and  subse- 
quent observation  has  shown  that  within  the  oospore  the  sex 
nuclei  are  alike  in  their  reaction  to  stains. 

Malfatti  (1891)  and  Lilienfeld  (1892-3)  claim  that  these 
reactions  are  dependent  upon  the  amount  of  nucleic  acid  present 
in  the  structures.  During  mitosis  the  chromosomes  consist  of 
nearly  pure  nucleic  acid  and  are  intensely  cyanophilous,  but  the 
protoplasm,  which  has  little  or  no  nucleic  acid,  is  erythrophi- 
lous.  There  is  a  gradual  transition  from  the  cyanophilous  con- 
dition to  the  erythrophilous,  and  vice  versa,  the  acid  structures 
taking  basic  stains  and  basic  structures  the  acid  stains. 

The  terms  "erythrophilous"  and  "cyanophilous"  are  falling 
into  disuse,  since  the  affinity  is  for  basic  and  acid  dyes,  rather  than 
for  blue  or  red  colors.  That  the  terms  are  misnomers  becomes 
evident  when  a  combination  like  safranin  (basic)  and  acid  green 
(acid)  is  used,  for  the  cyanophilous  structures  stain  red,  and  the 
erythrophilous  green. 

Probably  but  few  investigators  who  have  attained  any  pro- 
ficiency in  microtechnique  have  not  asked  themselves  how 
much  dependence  can  be  placed  upon  staining  reactions  as  a 
means  of  analysis.  Do  staining  reactions  enable  us  to  determine 
the  chemical  composition  of  a  structure  ?  If  two  structures  stain 
alike  with  Delafield's  haematoxylin,  does  this  mean  that  they 
have  the  same  chemical  composition ;  or  if,  on  the  other  hand, 
they  stain  differently,  must  they  necessarily  be  different  in  their 
chemical  composition?  Delafield's  haematoxylin,  when  care- 
fully used,  gives  a  rich  purple  color,  but  a  careful  examination 
will  often  show  that  in  the  same  preparation  some  structures 
stain  purple,  while  others  stain  red.  Does  this  mean  that  the 
purple  and  red  structures  must  have  a  different  chemical  com- 
position ?  Many  people  believe  that  structures  which  stain  dif- 
ferently with  a  given  stain  must  be  chemically  different,  but 
they  readily  agree  that  structures  which  stain  alike  are  not 
necessarily^  similar  in  chemical  composition.  Chromosomes  of 
dividing  nuclei  and  lignified  cell  walls  stain  alike  with  safranin ; 


50  Methods  in  Plant  Histology 

chromosomes  and  cellulose  cell  walls  stain  much  alike  with 
Delafield's  haematoxylin ;  but  everyone  recognizes  that  the 
chromosome  is  very  different  in  its  chemical  composition  from 
either  the  cellulose  or  the  lignified  wall. 

According  to  Fischer  (1897  and  1900),  stains  indicate  physi- 
cal but  not  chemical  composition.  Fischer  experimented  with 
substances  of  known  chemical  composition.  Egg  albumin  was 
shaken  until  small  granules  were  secured.  These  were  fixed 
with  the  usual  fixing  agents,  and  then  stained  with  Delafield's 
haematoxylin.  The  extremely  small  granules  stained  red,  while 
the  larger  ones  became  purple.  Since  the  granules  are  all  alike 
in  chemical  composition,  Fischer  concluded  that  the  difference 
in  staining  must  be  due  to  physical  differences.  With  safranin, 
followed  by  gentian  violet,  the  larger  granules  stain  red  and  the 
smaller  violet ;  if,  however,  the  gentian  violet  be  used  first,  then 
treated  with  acid  alcohol  and  followed  by  safranin,  the  larger 
granules  take  the  red  and  the  smaller  the  gentian  violet.  In 
root  tips  similar  results  were  obtained.  Safranin  followed  by 
gentian  violet  stained  chromosomes  red  and  spindle  fibers  violet, 
while  gentian  violet  followed  by  safranin  stained  the  chromo- 
somes violet  and  the  spindle  red.  One  often  reads  that  chro- 
mosomes owe  their  strong  staining  capacity  to  nuclein,  and 
especially  to  the  phosphorus,  but,  according  to  Fischer,  this  is 
shown  to  be  unfounded,  since  albumin  gives  similar  results  and 
yet  contains  no  phosphorus,  and  is  not  chemically  allied  to 
nuclein.  Delafield's  haematoxylin  is  one  of  the  so-called  nuclear 
stains.  The  nuclei  of  animals  and  plants  stain  deeply  with  this 
reagent,  but  cellulose  membranes,  the  dense  protoplasm  of 
embryonic  cells,  the  pyrenoids  of  green  algae,  and  many  other 
structures  resemble  nuclei  in  their  staining.  The  most  critical 
work  on  this  subject  has  been  done  by  those  who  are  investigat- 
ing the  structure  of  the  Cyanophyceae  and  Bacteria  to  determine 
whether  these  forms  have  nuclei  or  not.  Butschli  claims  that 
the  granules  which  stain  red  with  haematoxylin  are  to  be  identi- 
fied with  chromatin,  while  Fischer,  whose  results  have  just  been 
given,  claims  that  staining  indicates  merely  physical  differences. 


Ge?ieral  Remarks  on  Staining  51 

The  subject  cannot  yet  be  regarded  as  settled,  but  whatever  may 
be  true  in  regard  to  these  conflicting  theories,  all  agree  that 
stains  are  of  the  highest  importance  in  differentiating  struc- 
tures, and  in  bringing  out  details  which  would  otherwise  be 
invisible. 


CHAPTER  VIII. 

PRACTICAL  HINTS  ON  STAINING. 

In  later  chapters  specific  directions  will  be  given  for  making 
a  series  of  preparations  ranging  from  the  lowest  algae  to  the 
flowering  plants,  but  a  few  suggestions  will  be  made  here. 

The  number  of  stains  in  the  catalogues  is  becoming  so  great 
that  it  is  impossible  to  become  proficient  in  the  use  of  all  of 
the;m.  It  is  far  better  to  master  a  few  of  the  most  valuable  stains 
than  to  do  indifferent  work  with  many.  The  beginner,  especially 
if  rather  unacquainted  with  the  details  of  plant  structure,  may 
believe  that  he  has  an  excellent  preparation  when  it  is  really  a 
bad,  or  at  most  an  indifferent,  one.  To  illustrate,  let  us  suppose 
that  a  pollen  mother-cell  in  a  late  spirem  stage  has  been  stained 
with  cyanin  and  erythrosin.  A  preparation  in  which  the  cell 
merely  shows  a  differentiation  into  nucleus  and  cytoplasm  must 
be  classed  as  bad ;  if  the  nucleus  shows  a  definitely  outlined 
spirem  thread,  the  preparation  is  better,  but  is  still  only  indiffer- 
ent ;  if  the  thread  appears  as  a  delicate  red  ribbon  bordered  by 
blue  granules,  the  staining  may  be  regarded  as  a  success.  If 
mitotic  figures  have  been  stained  with  cyanin  and  erythrosin,  a 
first-class  preparation  should  show  blue  chromosomes  and  red 
spindles  ;  if  stained  with  safranin  and  gentian  violet,  the  chromo- 
somes should  be  red  and  the  spindles  violet. 

In  staining  growing  points,  apical  cells,  young  embryos, 
antheridia,  archegonia,  and  many  such  things,  the  cell  walls  are 
the  principal  things  to  be  differentiated,  if  the  preparations  are 
for  morphological  study.  As  a  rule,  it  is  better  in  such  cases  not 
to  use  double  staining,  but  to  select  a  stain  which  stains  the  cell 
walls  deeply  without  obscuring  them  by  staining  starch,  chloro- 
phyll, and  other  cell  contents.  For  example,  try  the  growing 
point  of  Equisetum  The  protoplasm  of  such  growing  points  is 
very  dense.     If  Delafield's  haematoxylin  and  erythrosin  be  used, 

S3 


54  Methods  in  Pla?it  Histology 

the  haematoxylin  will  stain  the  walls  and  nuclei,  and  will  slightly 
affect  the  other  cell  contents,  but  the  erythrosin  will  give  the 
cytoplasm  such  a  dense  stain  that  the  cell  walls  will  be  seriously 
obscured.  It  would  be  better  to  use  haematoxylin  alone.  The 
same  suggestion  may  well  be  observed  in  tracing  the  develop- 
ment of  antheridia,  archegonia,  embryos,  and  similar  structures. 
Permanent  preparations  are  an  absolute  necessity  for  the 
greater  part  of  most  advanced  work,  but  let  us  not  imagine  that 
we  cannot  examine  anything  until  we  have  made  a  permanent 
mount.  It  would  be  impossible  to  make  a  permanent  mount  of 
the  rotation  of  protoplasm.  It  is  better  for  many  purposes  to 
look  at  motile  spores  while  they  are  moving.  Use  Spirogyra 
while  it  is  fresh  and  green  (if  you  can),  and  use  permanent  prep- 
arations only  to  bring  out  nuclei  and  other  details  which  are 
not  so  easily  seen  in  living  material.  Examples  might  be  multi- 
plied. 


CHAPTER  IX. 

THE  CELLOIDIN  METHOD. 

"Celloidin  is  a  form  of  nitro-cellulose."  It  is  very  inflam- 
mable, but  does  not  explode.  It  may  be  obtained  in  the  form 
of  tablets  or  cuttings,  which  have  to  be  dissolved  in  a  mixture 
of  equal  parts  of  absolute  alcohol  and  ether.  It  is  customary  to 
use  two  solutions,  a  "thick"  and  a  "thin."  The  thick  solution 
(about  10  or  12  per  cent.)  should  have  about  the  consistency  of 
thick  syrup.  The  thin  may  be  made  by  mixing  equal  parts  of 
the  thick  and  ether  alcohol. 

As  mentioned  in  the  chapter  on  the  "General  Method,"  the 
killing,  washing,  and  dehydrating  are  the  same  as  for  the  paraffin 
method.  After  dehydrating  in  absolute  alcohol  the  succeeding 
steps  are  as  follows : 

1.  Ether  alcohol,  i  to  2  days. 

2.  Thin  celloidin,  2  to  6  days. 

3.  Thick  celloidin,  3  to  10  days. 

It  seems  better,  however,  to  begin  with  about  2  per  cent, 
celloidin  and  transfer  successively  through  4  per  cent.,  6  per 
cent.,  etc.,  to  12  per  cent.,  or  to  allow  the  2  per  cent,  to  concen- 
trate by  removing  the  cork  for  a  short  time  each  day. 

The  material  may  now  be  imbedded  and  mounted  upon 
a  block  at  the  same  time.  The  blocks  should  have  surface 
enough  to  accommodate  the  objects,  and  should  be  about  one- 
fourth  of  an  inch  thick.  White  pine  makes  good  blocks ;  cork 
is  much  inferior.  Place  the  block  for  a  moment  in  ether  alcohol 
and  then  dip  into  the  2  per  cent,  celloidin  the  end  of  the  block 
which  was  left  rough  by  the  saw.  With  the  forceps  remove  a 
piece  of  the  material  from  the  thick  celloidin  and  place  it  upon 
the  block,  taking  care  to  keep  it  right  side  up.  Dip  the  block 
with  its  object  first  in  thick  celloidin,  then  in  thin,  and  after 
exposing  to  the  air  for  a  few  minutes  drop  it  into  chloroform, 

55 


56  Methods  in  Plant  Histology 

where  it  should  remain  for  about  ten  to  twenty  hours.  It  should 
then  be  placed  in  equal  parts  of  glycerine  and  95  per  cent, 
alcohol,  where  it  may  be  kept  indefinitely.  If  the  material  is 
hard,  like  many  woody  stems,  it  will  cut  better  after  remaining 
in  this  mixture  for  a  couple  of  weeks. 

In  cutting,  the  knife  should  be  set  as  obliquely  as  possible, 
and  both  the  knife  and  the  object  should  be  kept  wet  with  the 
mixture  of  glycerine  and  alcohol.  As  fast  as  they  are  cut  the 
sections  are  transferred  with  a  soft  brush  to  70  per  cent,  alcohol. 
The  succeeding  steps  are  the  same  as  for  free-hand  sections,  but 
many  stains  are  not  available  because  they  stain  the  celloidin. 
Safranin  and  Delafield's  haematoxylin,  or  Delafield's  haematoxy- 
lin  and  eosin,  are  good  combinations  for  celloidin  sections.  Do 
not  use  absolute  alcohol  for  dehydrating,  since  it  dissolves  the 
celloidin,  but  transfer  from  95  per  cent,  alcohol  to  Eycleshymer's 
clearing  fluid  (equal  parts  of  bergamot  oil,  cedar  oil,  and  car- 
bolic acid),  which  clears  readily  from  95  per  cent,  alcohol. 
Mount  in  balsam. 

The  following  schedules  for  staining  celloidin  sections  will 
give  the  student  a  start.  The  times  given  will  vary  with  the 
thickness  of  the  section  and  character  of  the  tissue. 

a.   For  staining  in  Delafield's  haematoxylin  and  eosin  : 

1.  Seventy  per  cent.,  50  per  cent.,  and  35  per  cent,  alcohol,  2  to  5  minutes 
each. 

2.  Delafield's  haematoxylin,  5  to  30  minutes. 

3.  Wash  in  water,  5  minutes. 

4.  Thirty-five  per  cent,  and  50  per  cent,  alcohol,  2  to  5  minutes  each. 

5.  Acid  alcohol  (i  cc.  hydrochloric  acid -f-  100  cc.  of  70  per  cent,  alcohol) 
until  the  stain  is  extracted  from  the  celloidin,  or  at  least  until  the  celloidin 
retains  only  a  faint  pinkish  color. 

6.  Seventy  per  cent,  alcohol  (not  acid)  until  the  purple  color  replaces  the 
red  due  to  the  acid. 

7.  Eosin  (preferably  a  i  per  cent,  solution  in  70  per  cent,  alcohol),  2  to  5 
minutes. 

8.  Eighty-five  per  cent.,  95  per  cent,  alcohol,  2  to  5  minutes  each. 
Remember  that   100  per  cent,  alcohol  is  not  to  be  used  with  celloidin 


Celloidin  Method  57 

q.    Eycleshymer's  clearing  fluid  until  transparent,  usually  i  or  2  minutes, 

but  sometimes  5  or  10. 
10.    Mount  in  balsam. 

b.   For  staining  in  safranin  and  Delafield's  haematoxylin : 
.  I.    Seventy  per  cent.,  50  per  cent.,  and  35  per  cent,  alcohol,  2  to  5  minutes 
each, 

2.  Safranin,  6  to  24  hours,  preferably  the  longer  period. 

3.  Acid  alcohol  (try  about  i  drop  of  hydrochloric  acid  to  30  cc.  of  70  per 
cent,  alcohol)  until  the  stain  is  removed  from  the  celloidin  or  at  least 
becomes  very  faint. 

4.  Fifty  per  cent,  and  35  per  cent,  alcohol,  2  to  5  minutes  each. 

5.  Delafield's  haematoxylin,  2  to  5  minutes. 

6.  Wash  in  water,  5  minutes. 

7.  Thirty-five  per  cent,  and  50  per  cent,  alcohol,  2  to  5  minutes  each. 

8.  Acid  alcohol  (the  same  as  was  used  for  the  safranin)  until  the  stain  is 
extracted  from  the  celloidin.  If  it  is  found  that  this  double  immersion 
in  acid  alcohol  extracts  too  much  safranin,  the  third  step  may  be  omitted 
or  shortened  by  removing  the  sections  to  the  50  per  cent,  alcohol  before 
the  stain  is  thoroughly  removed  from  the  celloidin. 

g.    Eighty-five  per  cent,  and  95  per  cent,  alcohol,  2  to  5  minutes  each, 

10.  Eycleshymer's  clearing  fluid  until  cleared. 

1 1.  Mount  in  balsam. 

The  celloidin  method  has  its  disadvantages  as  well  as  its 
advantages.  It  is  extremely  slow  and  tedious,  and  it  is  rarely 
possible  to  cut  sections  thinner  than  lO/u,,  while,  on  the  other 
hand,  it  gives  smoother  sections.  The  entire  absence  of  heat 
makes  it  very  useful  for  delicate,  succulent  tissues.  Stems  and 
roots  which  cannot  be  handled  at  all  in  paraffin  cut  well  in 
celloidin,  and  much  larger  sections  can  be  cut  than  in  paraffin. 

When  material  is  to  be  imbedded,  use  celloidin  as  a  last 
resort.     Use  paraffin  when  you  ca7i,  celloidin  when  you  must. 

I  am  indebted  to  my  friend  Mr.  W.  B.  MacCallum  for  several 
suggestions  in  regard  to  this  method. 


CHAPTER   X. 


THE  GLYCERINE  METHOD. 


It  is  hard  to  get  the  filamentous  algae  and  fungi  into  balsam 
without  shrinking ;  consequently,  these  forms  are  usually  mounted 
in  glycerine  or  glycerine  jelly. 

Flemming's  fluid  and  chromo -acetic  acid  are  good  fixing 
agents.  Corrosive  sublimate  in  water,  or  in  70  per  cent,  alcohol, 
used  hot,  is  also  to  be  recommended.  For  general  morphology, 
stain  for  six  hours  or  over  night  in  a  ^  per  cent,  aqueous  solution 
of  eosin,  transfer  directly  to  a  i  per  cent,  solution  of  acetic  acid 
in  distilled  water,  and  allow  it  to  act  for  about  five  minutes  ;  wash 
thoroughly  in  water  to  remove  the  acid,  and  then  put  the 
material  into  a  watch-glass  in  a  10  percent,  solution  of  glycerine 
in  water.  The  watch-glass  should  be  kept  as  free  from  dust  as 
possible,  but  should  not  be  covered.  As  soon  as  the  solution 
appears  to  be  about  as  thick  as  pure  glycerine,  the  material  is 
ready  for  mounting.  Place  a  small  quantity  of  the  material  on 
a  slide,  arrange  it  carefully,  add  a  small  drop  of  glycerine,  and 
a  round  cover.  Seal  with  gold  size  (a  varnish  used  by  painters 
in  laying  gold  leaf).  None  of  the  sealing  media  will  stick  to 
moist  surfaces,  hence  it  is  essential  that  there  should  be  only 
enough  glycerine  to  come  to  the  edge  of  the  cover.  If  it  is 
desired  to  mount  rather  large  specimens,  like  the  antheridia  and 
oogonia  of  Chara,  it  is  best  to  spin  a  ring  on  the  slide,  thus 
forming  a  shallow  cell.  Before  the  ring  becomes  hard  the 
material  may  be  mounted  and  sealed,  or  the  ring  may  be  allowed 
to  harden,  and  just  before  a  mount  is  to  be  made  a  very  thin 
ring  of  the  varnish  may  be  added.  The  mount  is  firmer  if  the 
cover  is  not  only  sealed  in  the  usual  way,  but  also  sticks  to  the 
ring  underneath. 

If  glycerine  jelly  is  to  be  used,  place  the  bottle  in  warm 
59 


6o  Methods  i?i  Plant  Histology 

water  until  the  jelly  becomes  liquid,  but  avoid  any  unnecessary 
heat.  Take  the  material  from  the  glycerine,  add  a  drop  of  the 
warm  jelly,  and  seal  as  before.  More  detailed  directions  for 
mounting  material  in  glycerine  are  given  in  the  chapters  on  algae 
and  fungi. 


Part  II. 

In  the  preceding  chapters  the  principles  and  methods  of  tech- 
nique have  been  described  in  a  general  way.  It  is  often  diffi- 
cult, especially  for  a  beginner,  to  apply  general  principles  to 
specific  cases,  and,  besides,  the  types  which  he  might  select  for 
the  preparations  might  not  form  a  symmetrical  collection.  Con- 
sequently, a  series  of  forms  has  been  selected  which  will  not 
merely  serve  for  practice  in  microscopical  technique,  but  will 
also  furnish  the  student  with  preparations  for  a  fairly  satisfactory 
study  of  plant  structures  from  the  algae  up  to  the  angiosperms. 
It  is  not  at  all  our  purpose  to  discuss  general  morphology,  but 
rather  to  answer,  by  means  of  sketches  and  specific  directions, 
the  multitudinous  questions  which  confront  the  instructor  in  the 
laboratory.  For  those  who  have  had  a  thorough  training  in 
general  morphology  the  following  suggestions  will  be  in  some 
degree  surperfluous.  Those  who  are  beginning  the  study  of 
minute  plant  structure  are  referred  to  the  standard  text-books  for 
descriptions  of  the  plants  mentioned  here. 


STAi£ilOR11AL  SCHOOL, 


CHAPTER  XI. 


THALLOPHYTES.  ALG^. 
CYANOPHYCE-ffi. 
Wasserbliithe. — These  forms  occur  as  scums,  often  irides- 
cent, on  the  surface  of  stagnant  or  quiet  water.  Some  of  the 
commonest  forms  are  Ccelosphcerium  and  Anabcena  {jig.  ij). 
Some  of  the  Chlorophyceai  also 
occur  as  Wasserbliithe.  Where 
the  material  is  very  abundant, 
it  may  be  collected  by  simply 
skimming  it  off  with  a  wide- 
mouthed  bottle,  but  where  it  is 
rather  scarce,  it  is  better  to  filter 
the  water  through  a  cloth,  and 
finally  rinse  the  algse  off  into  a 
bottle.  Enough  formalin  may 
now  be  added  to  the  water  in 
the  bottle  to  make  a  2  per  cent, 
solution.  The  material  may  be 
kept  here  indefinitely,  but  after 
a  few  hours  it  is  ready  for  use. 
If  the  forms  are  small,  like 
Atiabce7ta,  smear  a  slide  lightly 
with  Mayer's  albumen  fixative, 
as  if  for  paraffin  sections,  add  a  drop  of  the  material  and  allow  it 
to  dry,  heat  the  slide  gently  to  coagulate  the  albumen,  or  immerse 
the  slide  in  strong  alcohol  for  a  few  minutes,  and  then  proceed 
with  the  staining.  Cyanin  and  erythrosin  is  a  good  combination 
for  differentiating  the  granules.  Delafield's  haematoxylin,  used 
alone,  stains  some  granules  purple  and  others  red.  Iron  alum- 
haematoxylin  is  excellent  for  heterocysts.  If  the  forms  are  large 
enough  to  collapse  with  such  treatment,  the  glycerine  method 

may  be  employed. 

63 


Coelosphaerium  Kuetzingianum.  B,  Anabaena 
flos-aquse.  C,  Anabaena  gigantea.  D  and  E,  a 
heterocyst  and  spore  of  A.  gigantea  drawn  from 
paraffin  sections  stained  in  cyanin  and  erythrosin. 


64 


Methods  in  Pla?it  Histology 


If  it  is  desirable  to  make  paraffin  sections,  put  the  material, 
drop  by  drop,  on  a  piece  of  blotting  paper  until  an  appreciable 
layer  of  sediment  is  obtained.  Get  the  paper  with  its  material 
into  paraffin  by  the  usual  method,  taking  great  care  not  to  wash 
the  algae  off.  After  imbedding,  trim  away  the 
paper  and  dip  the  block  in  melted  paraffin. 
Sections  can  now  be  cut  and  stained  in  the 
usual  manner. 

Oscillaria. — For  most  purposes  it  is  best  to 
study  Oscillaria  in  the  living  condition.  It  is 
readily  found  in  watering  troughs,  in  stagnant 
water,  on  damp  earth,  and  in  other  habitats. 
The  commonest  forms  have  a  deep  blue-green 
or  brownish  color.  For  the  purposes  of  identi- 
fication and  herbarium  specimens,  the  material 
may  simply  be  placed  on  a  slip  of  mica  and 
allowed  to  dry.  When  wanted  for  use,  add  a 
drop  of  water  and  a  cover,  and  the  mount  is  ready  for  examina- 
tion. For  sections  or 
for  glycerine  mounts  {\ 

fix    in    chromo-acetic 
acid.      {Fig-  ^4-) 

Rivularia. — This 
form  is  readily  found 
on  the  underside  of  the 
leaves  of  water-lilies 
[Nupkar,  NymphcBa, 
etc.)  ,  but  is  also  abun- 
dant on  submerged 
leaves  and  stems  of 
other   plants.     It    oc- 


FlG.  14.     Oscillaria. 

Portions  of  two  fila- 
ments, the  one  at  the 
right  showing  a  hormo- 
gonium,  h. 


Fig.  15. 
A.  nodule   crushed  under 


glass.     B,  tour  filaments  more 


curs     in     the     form     of    Wghly  magnified,  showing  heterocysts  at  the  base. 

translucent,  gelatinous  nodules  of  various  sizes.  Chromo-acetic 
acid  gives  beautiful  preparations,  but  good  results  can  also  be 
secured  from  formalin  or  picric-acid  material. 

The  most  instructive  preparations  for  morphological  study 


Thallophytes.     AlgcB 


65 


Fig.  16.    Tolypothrix. 
b,  a  false    branch,     h,  hetero- 


can  be  obtained  by  the  glycerine  method. 
Stain  in  eosin  or  Mayer's  haem-alum.  When 
ready  for  mounting,  crush  a  small  nodule 
by  pressing  on  the  cover-glass.  Fig.  i§  is 
drawn  from  such  a  preparation.  The  par- 
affin method  is  easily  applied,  since  the 
gelatinous  matrix  keeps  the  plants  in 
place.  Glceotrichia,  Nostoc,  and  forms  of 
similar  habit  may  be  prepared  in  the  same 
way. 

Tolypothrix. — This  form  occurs  as  small 
tufts,  either  floating  in  stagnant  water  or 
attached  to  plants  and  stones.  It  furnishes 
an  excellent  example  of  false  branching. 
{Fig.  16.)  Scytojiema  is  a  similar  form 
which  is  fairly  common.  The  glycerine 
method  should  be  employed  for  perma- 
nent preparations,  but  this,  like  all  small  '^^^' 
filamentous  algae,  may  be  dried  on  mica  for  herbarium  pur- 
poses. 

CHLOROPHYCEiE. 

The  ponds,  ditches,  and  rivers  of  any  locality  will  yield  an 
abundance  and  variety  both  of  the  unicellular  and  multicel- 
lular members  of  this  group.  The  unicellular  and  filamentous 
members,  together  with  such  forms  as  Volvox,  are  best  prepared 
by  the  glycerine  method.  The  structure  is  so  much  more  com- 
plicated than  in  the  Cyanophyceae  that  it  demands  far  more  care 
and  skill  to  make  good  preparations,  Chromo-acetic  acid  is  a 
good  killing  and  fixing  agent  for  the  whole  group,  but  Flem- 
ming's  fluid  (weaker  solution)  seems  to  be  a  little  better  in  some 
instances.  Very  good  results  have  been  obtained  by  adding 
about  5  cc.  of  I  per  cent,  osmic  acid  to  100  cc.  of  chromo- 
acetic  acid  (Schaffner's  formula).  A  formula  which  gives  satis- 
factory results  with  Spirogyra  may  cause  plasmolysis  with 
Cladophora.  The  given  filament  should  be  placed  under  the 
microscope  in  the  fixing  agent,  and,  if  plasmolysis  occurs,  the 


66 


Methods  in  Plant  Histology 


Fig.  17. 
A ,  Vaucheria  geminata. 


B 


Vaucheria. 
^.V.sessilis. 


chromic  should  be  weakened  or  the  acetic  strengthened  until  the 
suitable  proportions  are  determined.  This  is  a  slow  process,  but 
Cladophora  and  Vaucheria  are  almost  sure  to  shrink  without  it. 
About  twenty-four  hours  in  any  of  the  chromic  series  and  a  four 
to  ten  hours  washing  in  water  will  be.  sufficient  for  members  of 

this   group.      Only  a   few 
o       of  the  most  familiar  forms 
will  be  mentioned. 

Vaucheria. — This  form 
can  always  be  obtained  in 
greenhouses,  especially  in 
the  fernery,  where  it  forms 
3         a  green  felt  on  the  pots. 
'''*^^°'"'-  The    greenhouse    form    is 

likely  to  be  Vaucheria  sessilis.  Another  species,  V.  geminata,  is 
very  common  in  the  spring,  when  it  may  be  found  in  ponds  and 
ditches.  {^Fig.  ly.^  It  is  extremely  difficult  to  get  mounts  show- 
ing the  nuclei.     The  following  method  is  sometimes  successful : 

1.  Chromo-acetic  acid  (Schaffner's  formula),  24  hours. 

2.  Wash  in  water,  4  to  10  hours. 

3.  Iron  alum,  2  to  4  hours. 

4.  Water,  15  to  30  minutes. 

5.  One-half  per  cent,  haematoxylin,  over  night. 

6.  Wash  in  water,  5  to  ro  minutes. 

7.  Iron  alum  until  details  become  clear.    This  may  take  only  a  few  minutes, 
but  may  take  an  hour. 

8.  Wash  thoroughly  in  water,  i  to  4  hours. 

g.    Ten  per  cent,  glycerine  and  allow  the  glycerine  to  thicken. 
10.    Mount  and  seal. 

Cladophora. — This  is  found  attached  to  sticks  and  stones  in 
quiet  or  running  water.  It  is  easily  recognized  by  its  character- 
istic branching.  {^Fig.  18.^  The  nuclei  of  the  ccenocytic  seg- 
ments are  readily  brought  out  by  the  method  just  described  for 
Vaucheria.  Alum  carmine  and  Mayer's  haem-alum  are  also  good 
stains  for  the  nuclei. 

Hydrodictyon. — This  is  popularly  known  as  the  "water-net." 
Nets  of  all  sizes  should  be  selected  for  study.     The  segments 


Thallophytes.     Algce 


67 


are  coenocytic,  and  the  nuclei  are  hard  to  differentiate  except  in 
the  younger  segments.  The  method  given  for  Cladophora  yields 
good  results.  The  young  nets  forming  within  the  older  seg- 
ments are  easily  demonstrated  by  Mayer's  haem-alum.  Paraffin 
sections  stained  in  cyanin  and  erythrosin,  iron 
alum-hgematoxylin,  or  the  safranin-gentian  violet- 
orange  combinationwill  repay  the  trouble,  especially 
in  case  of  the  oldest  segments  within  which  new 
nets  are  forming.  The  habit  is  beautifully  shown 
in  preparations  stained  with  eosin.  The  eosin  ( i  per 
cent,  aqueous  solution)  should  act  for  about  twenty- 
four  hours.  Then  transfer  to  i  per  cent,  acetic 
acid  for  a  few  minutes.  If  the  stain  comes  out 
rapidly  in  the  acid,  one  minute  may  be  sufficient, 
but  if  the  stain  does  not  wash  out,  it  is  better  to 
let  the  acid  act  for  four  or  five  minutes.  Wash 
thoroughly  in  water  to  remove  all  trace  of  acid, 
or  the  preparation  will 
fade.  Transfer  to  10  per 
cent,  glycerine  and  pro-      Fixed. in  chromo- 

di  /  7-"'  \      acetic  acid,  Stained  in 

as  usual.       [Ftg.ig.)     HaidenhaiVs     iron 
_     .  rr^,    .  .        alum-h3ematoxylin. 

Spirogyra.  —  This  is 
probably  the  most  widely  known  of  all 
the  algae,  and,  fortunately,  it  is  rather 
easy  to  obtain  beautiful  and  instructive 
preparations.  The  following  is  a  good 
fixing  agent  for  most  Spirogyras; 

Chromic  acid,  -^^  g. 

Glacial  acetic  acid,  -i\  g. 

Water,  99  cc. 

The  addition  of  i  cc.  of  i  per  cent,  osmic  acid  seems  to 
improve  it  without  causing  any  blackening.  Flemming's  weaker 
solution  is  excellent.  If  it  causes  too  much  blackening,  as  it 
probably  will,  the  material,  after  being  washed,  should  be  placed 
in  weak  peroxide  of  hydrogen  (one  part  H^Oa  to  three  parts 
H2O)  until  the  blackening  due  to  osmic  acid  disappears.     After 


Fig 


Cladophora. 


Fig.    19.     Hydrodictyon. 
A  small  portion  of  a  young  net 


68 


Methods  i?i  Plant  Histology 


a  moment's  washing  in  water  it  is  then  ready  for  the  stain.  The 
iron  alum-haematoxylin,  as  just  described,  brings  out  the  nuclei 
and  pyrenoids  with  great  distinctness,  {^Fig.  20.)  A  few  min- 
utes in  aqueous  eosin  after  the  last  washing  in  water  often  gives 
a  beautiful   differentiation,   but   the    preparations   will    be  quite 


Fig.  20.     Spirogyra. 

From  material  fixed  in  chromo- acetic  acid  and  stained  in  iron  alum-haematoxylin.     A 
single  cell  showing  nucleus,  chromatophore,  and  pyrenoids 


division.     C, ; 


,  a  nucleus  undergoing 
ng  nucleus,     D,  zygospores,  each  showing  two  nuclei. 


inferior  if  the  eosin  is  allowed  to  stain  too  deeply,     Mayer's 
haem-alum  is  a  better  stain  for  stages  in  conjugation. 

It  is  difficult  to  get  Spirogyra  into  paraffin  without  shrinking, 
.but  it  can*be  done.     Watch  carefully  and  note  where  plasmolysis 


Thallophytes.     Algm 


69 


occurs.  There  will  probably  be  little  or  no  trouble  until 
the  transfer  from  100  per  cent,  alcohol  to  the  clearing  agent. 
Make  this  transfer  as  gradual  as  may  be  necessary.  After  the 
pure  xylol  or  other  clearing  agent  is  reached,  add  a  lump  of 
paraffin  large  enough  to  saturate  the  clearing  fluid  at  a  tempera- 
ture of  40  to  45°  C.  Allow  the  xylol  to  evaporate  at  this 
temperature  and  imbed  as  usual,  taking  care  to 
keep  the  filaments  as  nearly  parallel  as  possible. 
Many  elegant  combinations,  like  cyanin  and 
erythrosin,  fuchsin  and  iodine  green,  safranin- 
gentian  violet-orange,  and  others  not  available 
for  glycerine  preparations,  can  be  used  with 
paraffin  sections.  It  is  comparatively  easy  to 
get  any  such  alga  into  celloidin.  Safranin  and 
Delafield's  haematoxylin  then  make  a  good 
combination. 

Zygnema — Use  the  same  methods  as  for 
Spirogyra.  In  staining  conjugating  material  the 
stain  should  be  extracted  until  the  four  chroma- 
tophores  of  the  zygospore  become  distinct.  The 
nuclei  are  comparatively  small  and  unsatisfac- 

.  #-!-.,  ,     11     ,  1  ,1  11         The  filament  at   the  left 

tory.      Ihe   stellate   chromatophores   are  well  shows  three  zygospores  and 

,  ,  ,  ,  .  -t  r    •  1  0"C  parthenogenetic  spore 

brought  out  by  alum  carmme.     If  iron  alum-  which  is  distinguished  by 

having  only  two  chromato- 

haematoxylin  and  eosin  are  used,  the  eosin  may  phores.  The  fiiamem  on  the 

•'  •'      right  shows  two  cells,  each 

well  be  much  deeper  than  in  case  of  Spirogyra.  with  a  pair  of  steiiate  chro- 

1  c        oy  matophores.     Drawn    from 

(pier    2T\  material  fixed  in  2  per  cent. 

\J.  i-^ .    ^i.j  formalin  and  stained  in  iron 

Diatoms.— Diatoms  and  desmids  have  been  -'""^h^-^-'o-y'*"- 
variously  classified,  and  their  position  is  not  yet  fully  determined. 
Living  diatoms  are  often  found  clinging  in  great  numbers  to  fila- 
mentous algse,  or  forming  gelatinous  masses  on  various  submerged 
plants.  It  is  difficult  to  get  really  good  preparations  showing  the 
nucleus  and  chromatophores.  If  the  diatoms  are  clinging  to  fila- 
mentous algae,  the  algae  with  the  diatoms  attached  may  be  put 
into  chromo-acetic  acid  (twenty-four  hours),  washed  in  water, 
stained,  passed  up  through  the  alcohols,  and  cleared  in  xylol, 
or,  better,  in  clove  oil  or  bergamot  oil,  which  do   not  dry  up  so 


Fig.  21.     Zygnema. 


70 


Methods  in  Plant  Histology 


rapidly.  Here  the  diatoms  may  be  picked  or  scraped  off  from 
the  other  algae,  which  will  probably  have  become  much  shrunken 
by  this  treatment.  Mount  in  balsam.  Haidenhain's  iron  alum- 
haematoxylin  is  recommended  for  the  nucleus  and  the  centro- 
some,  which  is  quite  prominent  in  diatoms.  Delafield's  haema- 
toxylin   and   erythrosin   give  a   good  view  of  the  nucleus  and 

chromatophore.  If  a  glycer- 
ine mount  is  preferred,  the 
iron  alum-haematoxylin  is  a 
good  stain. 

When  the  material  is  in 
gelatinous  masses,  it  may  be 
fixed  in  chromo-acetic  acid 
and  imbedded  in  paraffin. 
There  will,  of  course,  be  some 
difficulty  in  cutting,  and  many 
frustules  will  be  broken,  but 
there  will,  nevertheless,  be 
occasional  views  which  show 
details  better  than  when  the 
diatoms   are  mounted  whole. 

Lriceratmm  sp.  ThC   SlHcioUS   Shclls  Of  dia- 

toms  are  among  the  most  beautiful  objects  which  could  be  exam- 
ined with  the  microscope  {fig.  22) .  To  obtain  perfectly  clean 
mounts  requires  considerable  time  and  patience,  but  when  the 
^material  is  once  cleaned,  preparations  may  be  made  at  any  time 
with  very  little  trouble.  Diatom  enthusiasts  have  devised  numer- 
ous methods  for  cleaning  diatoms,  and  separating  the  various 
forms  from  each  other,  but  we  shall  give  here  only  a  few  simple, 
practical  methods. 

Material  for  mounts  of  frustules  of  living  forms  "  may  be 
obtained  by  skimming  off  the  brownish  scum  found  on  ponds, 
by  squeezing  out  water  weeds,  by  scraping  sticks  and  stones 
which  are  covered  at  high  water,  or  from  the  mud  of  filter  beds 
at  pumping  works,  or  in  other  places.  The  material  is  put  in  a 
dish  of  water,  and  after  it  has  settled  the  water  is  decanted. 


Diatoms.     X  255- 

A^  Pleurosigma  angulatum.  B,  Navicula  dactylis. 
C,  Synedra  biceps.  D,  Gomphonema  sphaerophorum. 
E,'tx\(. 


Thallophytes.     Algce  yi 

This  is  repeated  until  the  water  will  clear  in  about  one-half  hour.- 
The  sediment  is  then  treated  with  an  equal  bulk  of  sulphuric 
acid,  after  which  bichromate  of  potash  is  added  until  all  action 
ceases.  .After  a  couple  of  hours  the  acid  is  washed  out.  To 
separate  the  diatoms,  place  the  sediment  in  a  glass  dish  with 
water,  and  when  the  water  becomes  clear  give  the  dish  a  slight 
rotary  motion.  This  will  bring  the  diatoms  to  the  top,  when 
they  may  be  removed  with  a  pipette  and  placed  in  alcohol.  To 
mount,  place  a  number  in  distilled  water,  evaporate  a  few  drops 
of  the  mixture  on  a  cover-glass,  which  is  then  mounted  on  a 
slide  in  balsam."  (From  a  review  of  Dr.  Wood's  paper  on 
"  Diatoms," /^«^-  App.  Mic,  March,  1899.) 

Many  scouring  soaps  and  silver  polishes  contain  large  quan- 
tities of  fossil  diatoms,  and  the  diatomaceous  earths  are  particu- 
larly rich.  Break  up  a  small  lump  of  such  material  and  boil  it 
in  hydrochloric  acid.  A  test-tube  is  very  convenient  for  this 
process.  Let  the  diatoms  settle,  pour  off  the  acid,  and  then 
wash  in  water.  As  soon  as  the  diatoms  settle,  the  water  should 
be  poured  off.  The  washing  should  be  continued  until  the 
hydrochloric  acid  has  been  removed.  When  the  washing  is 
complete,  pour  on  a  little  absolute  alcohol,  and  after  a  few 
minutes  pour  off  the  alcohol  and  add  equal  parts  of  turpentine 
and  carbolic  acid.  The  material  will  keep  indefinitely  in  this 
condition  and  may  be  mounted  in  balsam  at  any  time.  In  mak- 
ing a  mount,  put  a  little  of  the  material  on  a  slide  and  allow  it 
to  become  dry,  or  nearly  dry,  and  then  add  the  balsam  and  cover. 
If  the  balsam  should  be  added  too  soon,  the  diatoms  are  likely 
to  move  to  the  edge  of  the  cover. 

Desmids. — When  these  forms  are  very  abundant,  they  may  be 
treated  like  the  filamentous  algs,  except  that  extreme  care  must 
be  taken  lest  the  desmids  be  lost  while  changing  fluids.  It 
often  happens  that  a  single  desirable  desmid  appears  when 
examining  field  collections.  In  such  a  case,  remove  it  with  a 
fine  pipette,  and  get  it  into  a  drop  of  water  on  a  clean  slide, 
invert  it  over  a  bottle  of  i  per  cent,  osmic  acid  for  a  minute, 
leave  the  slide  exposed  to  the  air  until  the  water  has  almost  all 


72 


Methods  in  Plant  Histology 


the 


Evaporated,  and  then  add  a  drop  of  lo  per  cent,  glycerine.  In 
a  few  hours  (six  to  twenty-four)  put  on  a  cover  and  seal.  It 
requires  more  time,  care,  and  patience  than  it  is  worth  to 
attempt  staining  in  such  a  case.      {^Fig.  2j.) 

Oedogonium. —  In  selecting  material  it  will  be  better  for  teach- 
ing purposes  to  choose  the  larger  monoecious  forms.  The  nuclei, 
pyrenoids,  and  chromatophores  are 
easily  differentiated.  Mayer's  haem- 
alum  is  a  good  stain,  especially  for 
the  antheridia.  Alum  carmine  or 
eosin  will  bring  out 
{Fig.  24.) 

Chara. — This  form 
is  so  large  and  coarse 
that  it  hardly  pays  to 
mount  it  in  glycerine. 
If  a  glycerine  mount  is  ^y. 
desired  to  show  the  an- 
theridia and  oogonia  in 
Fig.  23.   Desmids.   X  255.  positiou,  Spin  a  ring  of 

From  glycerine  preparations.  Not  stained. 
A,   Cosmarium   pectinoides.     B,    Closte-     CemCnt      On       thc       slidc, 
rium  cucumis.     C,  Staurastrum  comutum. 
A  Arthrodesmus  octocornis.  thuS    making     3.    Ccll     in 

which  small  portions  of  the  plant  may  be  mounted. 
For  parafifin  sections  select  the  tip  of  the  plant, 
a  piece  about  half  an  inch  in  length.  Sections  of 
this  may  show,  not  only  the  large  apical  cell,  but 
also  various  stages  in  the  development  of  antheri-  „         r,  j 

°  c  Fig.  24.  Oedogonium 

dia  and    oogonia.     Delafield's    haematoxylin  is  a       "oduiosum. 

,  .         .  ,  .-  ,,  1     r  1  '^>    antheridia.      0, 

very  good   stam  for  the  apical   cell   and  for  the  oogonium.  Drawn 

.  .  rr^,  horn  material  fixed  in 

development  of  antheridia  and  oogonia.    The  later  i  per  cent,  chromic 

^  "  acid,  and   stained  in 

stages  in  the  development  of  antherozoids  are  Mayer's hsem-aium. 
brought  out  more  clearly  by  the  safranin-gentian  violet-orange, 
or  by  cyanin  and  erythrosin,  but  here  unusual  care  must  be  taken 
not  to  stain  too  deeply. 

Good  preparations  showing  shield,  manubrium,  capitula,  and 
filaments  may  be  obtained   by  staining  in  bulk  in  alum  carmine 


Thallophytes.     AlgcB 


73 


and  then  crushing  the  antheridium  under  the  cover-glass  after 
the  specimen  is  in  balsam.      {^Fig.  2j.) 

PHJEOPHYCEiE. 

The  brown  algae  are  almost  exclusively  marine.     The  slime, 
so  prevalent  in  the  group,  often  makes  the  technique  difficult. 

Ectocarpus. —  Fix  in 
chromo-acetic  acid 
(twenty-four  hours), 
wash  in  fresh  water, 
since  the  salt  of  sea 
water  may  cause  incon- 
venience in  subsequent 
processes.  Stock  mate- 
rial should  be  passed  up 


A,  portion  of  a  branch  showing  an  antheridium,  a,  and  an 
oogonium,  o.  B,  median  longitudinal  section  of  an  apical  cell. 
Drawn  from  a  preparation  fixed  in  chromo-acetic  acid,  and 
stained  in  Delafield's  haematoxylin. 

to  70  per  cent,  alcohol  for  safe  keeping. 
Eosin  or  Mayer's  haem-alum  are  good  for 
glycerine  mounts.  If  paraffin  sections  are 
to  be  made,  the  material  must  be  brought 
very  gradually  from  absolute  alcohol  into 
the  clearing  agent,  and  from  the  clearing 
agent  into  the  paraffin.      {^Fig.  26.) 

Other  filamentous  members  of  the 
group,  as  well  as  the  more  delicate  mem- 
branous form.s,  may  be  treated  like  Ecto- 
carpus. 

Fucus. — Fucus  may  be  fixed,  washed,  and  preserved  like 
Ectocarpus.  It  is  difficult  to  get  paraffin  sections  across  the  whole 
fertile  branch,  but  elegant  sections  may  be  obtained  by  cutting 


Fig.  26.     Ectocarpus  confervoides. 

From  a  preparation  stained  in 
Mayer's  haem-alum,  and  mounted  in 
glycerine.  X  255.  in,  multilocu- 
lar  sporangium,     w,  unilocular  spo- 


74 


Methods  in  Pla?it  Histology 


narrow  strips  containing  a  few  conceptacles.  The  safranin- 
gentian  violet-orange  combination  is  good  for  such  sections.  For 
such  views  as  are  represented  in  fig.  2y,  C  and  D,  the  material 

should  be  stained 
in  bulk  in  borax 
carmine  or  alum 
carmine.  The 
process  for  borax 
carmine  is  as  fol- 
lows : 
[.    Borax  carmine,  24 

hours. 
3.  Acid  alcohol  (2 
drops  of  HCl  in 
50  cc.  of  70  per 
eent.alcohol),until 
the  color  becomes 
a  clear  red.  This 
may  take  an  hour 
or  even  a  day. 

3.  Seventy  to  loo  per 
cent,  alcohol,  2 
hours  each. 

4.  Clear  in  cedar  oil, 
bergamot  oil,  or 
oil  of  cloves. 

5.  Tease  out  the  con- 
tents of  the  con- 
ceptacles suffi- 
ciently to  show 
details,  and  mount 
in  balsam. 


Fig.  27.    Fucus  vesiculosus. 


A,  small  portion  of  plant  showing  bladders  and  fruiting  branches. 
One-half  natural  size.  B,  transverse  section  of  fruiting  branch  showing 
oogonial  conceptacles.  X  6.  C,  antheridia  and  paraphyses.  From  a 
preparation  fixed  in  chrorao- acetic  acid,  stained  in  borax  carmine,  teased 
out  and  mounted  in  balsam.  X  255.  D,  oogonium  showing  five  of  the 
eight  oospheres.     Prepared  as  in  C. 


The  process  for  alum  carmine  is  the  same,  except  that  no 
acid  alcohol  is  used. 

Sections  like  that  shown  in  B  are  easily  cut  in  celloidin. 
After  staining  in  borax  carmine  or  alum  carmine,  imbed  in 
celloidin  in  the  usual  way.  After  hardening  the  celloidin  in 
chloroform,  put  the  block  into  95  per  cent,  alcohol  for  two  or 
three  hours,  and  then  into  Eycleshymer's  clearing  fluid  (equal 
parts  bergamot  oil,  cedar  oil,  and  carbolic  acid),  until  thoroughly 
cleared.     The  block  may  be  left  here  indefinitely,  and  sections 


Thallophytes.     AlgcB 


75 


B 


may  at  any  time  be  mounted  in  balsam  as  soon  as  they  are 
cut. 

Chorda,  Lamifiaria,  and  similar  forms  may  be  treated  like  Fucus. 

RHODOPHYCEiE. 

The  red  algae  belong  almost  exclusively  to  salt  water,  -but  a 
few  genera  are  found  only  in  fresh  water,  usually  in  running  water, 
and  a  few  forms  occur  both        ^ 
in  salt  and  in  fresh  water. 

The    technique    is    more 
difficult  than  in  the  case  of 
the  brown  algae.    Until  some- 
thing better  is  suggested,  the   p 
same  method  of  fixing  and 
washing  may  be  used  as  for 
the  brown  algae.     Picric  acid, 
corrosive  sublimate,  and  ab-     / 
solute  alcohol    have    been 
tried,  but  do  not  give  as  good 
results  as  the  chromo-acetic   ^ 
acid  or  Flemming's  fluid. 

Batrachospermum. — This 
is  a  green,  fresh-water  mem- 
ber of  the  red  algae.  It  is 
not  very  uncommon  in  small 
streams.      {Fig.  28.) 

TViP-     rf^llc      ar<^      cr.     email  branches  and  several  cystocarps.     X  25.    ^,  a  procarpib 

i  HC     CCllb      die      bU     Sllldll  branch   showing   carpogonium   (/),  and  trichogyne  (i'), 

.1       ,     .,     •       1  11  ,1  1-1  with  an  antherozoid  (j)  attached.     X  255.     C,  a  younger 

tnat   It   is    naraly   WOrtn   Wnlle  branch   showing   carpogonlum   and  trichogyne.     X  255. 

...  ,  ,  D,  branch  with  three  antherozoids.     X  255. 

to  attempt  sectionmg  them. 

Very  good  preparations  showing  the  nuclei  may  be  obtained  by 
staining  in  Mayer's  hsem-alum,  or  Haidenhain's  iron  alum-haema- 
toxylin.  After  the  material  is  in  glycerine  ready  for  mounting, 
tease  out  a  small  portion,  and  still  further  dissociate  the  filaments 
by  tapping  smartly  on  the  cover. 

Material  stained  in  eosin  shows  the  external  structure  well, 
but  may  not  bring  out  the  nuclei. 


Fig.  28,     Batrachospermum  moniliforme. 

From  a  preparation  stained  in  Mayer's  haem-alum  and 
mounted   in   glycerine.      A,  portion    of    plant    showing 


76 


Methods  in  Plant  Histology 


Polysiphonia. —  For  preparations  like  those  shown  in  fig.  2g 
eosin  is  a  very  good  stain.      To  get  a  brilliant  coloring,  stain 

for  about  twenty-four  hours, 
so  that  the  i  per  cent,  acetic 
acid  may  be  allowed  to  act 
for  several  minutes  without 
making  the  stain  look  weak. 
Wash  thoroughly  in  water. 
Not  the  slightest  trace  of 
color  should  be  allowed  to 
come  out  in  the  glycerine. 

Sections  showing  the  cen- 
tral and  peripheral  siphons 
and  other  gross  features  are 
easily  cut  in  celloidin.  It  is 
not  very  difficult  to  cut  par- 
affin sections,  but  the  nuclei 
are  so  small  and  so  hard  to 
bring  out  that  such  prepa- 
rations had  better  be  left  for  the  specialist. 


Fig.  29.     Polysiphonia  fibrillosa. 

From  a  preparation  fixed  in  chromo- acetic  acid,  stained 
in  eosin.  and  mounted  in  glycerine.  X  255.  A,  an 
antheridium.  B,  a  cystocarp  with  carpospores.  C, 
tetrasporic  branch  with  tetraspores. 


CHAPTER  XIL 

THALLOPHYTES.     FUNGI. 

SCHIZOMYCETES. 
Bacteria. —  The  methods  of  modern  bacteriological  technique 
are  so  numerous  and  so  specialized  that  we  must  refer  to  laboratory 
manuals  for  instruction  in  this  subject.     The  method  given  here 
will  merely  enable  the  student  to  study 
the   form   and   size   of    those   bacteria  d't^^'^i 


which  are  more  easily  demonstrated.  ''^^x-'  ■^■^^*^|^"' 

Foul  water  at  the  outlets  of  sewers  /  >^       ^  •^      so-    '^^. 

and  such  places  will  usually  afford  an  X.  4.     "^    "^     ^)^K 

abundance   of    coccus,  bacillus,  spiril-  f^^  \  ®    ^'''    ^^ 


•  R         "^JJ"  C 


lum,  and  beggiatoa  forms.  Place  a 
drop  of  the  water  on  a  slide,  heat  it 
gently  until  the  water  evaporates,  then 

stain   with    fuchsin    or    methyl    violet,      f^^"^  ^^^k^ 

dehydrate,   clear  in   xylol,  and  mount 

in  balsam  {fig.  so)-  C     3   ^"-J/   "    C 

Fine  preparations  may  be  obtained  fig.  30.  Bacteria.   X535. 

by     inoculating    a    mouse    with     Anthrax  ^.Badllusanthracis,  from  a  paraffin 

•'  "  section  cut  from  the  liver  of  a  mouse. 

or  some  other  form,  and  then  cutting  ^^^'^^ZT^^^^^^'^ 
paraffin  sections  of  favorable  organs.  cTsTolin^str^uV^Ffor a^S-^ 
Gentian    violet    with    a   faint   bismark     SumTp'  'Frfm"T;rS:ratio''n 

1  r  i_        1  11  1        stained  in  fuchsin. 

brown  tor  a  background  makes  a  good 

combination.     The  following  schedule  gives  good  results  with 

Anthrax  and  many  other  bacteria : 

1.  Gentian  violet,  5  minutes. 

2.  Rinse  in  water  a  few  seconds. 

3.  Gram's  solution  (iodine  1  gram,  potassium  iodide  2  grams,  water  300  cc.) 
until  the  color  is  almost  or  quite  black  ;  this  will  generally  require  i  or 
2  minutes. 

4.  Ninety-five  per  cent,  alcohol  until  the  color  has  nearly  disappeared. 

77 


78  Methods  in  Plant  Histology 

5.  Rinse  in  water  and  examine.     If  the  bacteria  are  well  stained,  a  counter- 
stain  for  the  background  may  be  added. 

6.  Erythrosin,  3  or  4  seconds ;  or  bismark  brown  5  or  10  seconds. 

7.  Ninety-five  and  100  per  cent,  alcohol,  dehydrating  as  rapidly  as  possible. 
Not  more  than  5  or  10  seconds  can  usually  be  allowed. 

8.  Xylol. 

9.  Balsam. 

Leptothrix  may  often  be  obtained  by  scraping  the  inside  of 
the  cheek.  Beggiatoa,  a  form  with  oscillating  movements  like 
Oscillaria,  is  often  found  in  foul  water.  Its  presence  may  be 
indicated  by  whitish  patches  on  the  bottom. 

It  is  doubtful  whether  the  bacteria  possess  even  a  morpho- 
logical forerunner  of  the  nucleus  of  higher  plants,  consequently 
there  need  be  no  disappointment  if  the  larger  forms,  like  some 
of  the  Beggiatoas,  fail  to  show  a  nucleus. 

MYXOMYCETES. 

With  the  exception  of  a  few  forms  like  Fuligo  (often  found 
on  oak  stumps  and  on  oak  bark  in  tan  yards),  the  Myxomycetes 
are  small,  and  are  usually  overlooked  by  collectors.    ^Fig.ji.)    A 

careful  examination  of  rotting  logs 
in  moist  woods  will  usually  reveal 
an  abundance  of  these  delicate 
and  beautiful  organisms.  They 
should  be  pinned  to  the  bottom 
of  a  box  for  safe  carrying.  For 
herbarium  specimens  they  are 
simply  allowed  to  dry,  and  are 
C  then  fastened  with  glue  or  paste 

Myxomycetes.  ^^  ^j^^  bottOm   of    a  Small  boX. 

Growing  on  rotten  wood,    /i ,  Hemitrichia  rubi- 

formis.   X  20.  B,  Stemonitis  ferruginea.  Nat-  Plasmodia  and  voung  SDoran- 

ural  size.     C,  Trichia  varia.     Xi%.  .  .  . 

gia  may  be  fixed  in  chromo-acetic 
acid  or  Flemming's  fluid.  Sections  are  easily  cut  in  paraffin, 
and  should  not  be  more  than  5  ft  in  thickness,  and  should  be 
thinner,  if  possible.  Acid  fuchsin  and  iodine  green  is  a  good 
stain.  Delafield's  haematoxylin  used  alone  or  with  a  little 
orange  G.  is  also  to  be  recommended.   ,  Excellent  methods  for 


Thallophytes.     Fungi 


79 


living  cultures  were  given  in  the  January  and   February  (i 
numbers  of  1\\q  Jour?ial  of  Applied  Microscopy. 


PHYCOMYCETES. 

Mucor. — This  familiar  mold  appears  with  great  regularity  on 
bread.     The  following  is  a  sure  and  rapid  method  for  obtaining 
Mucor:     Place  a  glass  tumbler  in  a  plate  of  water,  put  on  the 
tumbler   a  slice   of    bread   which 
has  been  exposed  to  the  air  for  a 
day,  and  cover  with  a  glass  jar. 

To  obtain  such  a  series  as  is 
shown  in  A-D  of  fig.  J2,  the  ma- 
terial should  be  studied  before 
the  sporangia  begin  to  turn  black. 
The  phase  in  the  life-history  indi- 
cated in  F-// is  rarely  seen.  The 
writer  would  be  glad  to  hear  from 
any  who  have  met  this  phase, 
especially  if  the  information  could 
be  accompanied  by  a  few  zygo- 
spores. 

Ci    ,  .           .           /  A-D,   successive  stages  in  the  development 

OrrOSlVe     sublimate      (^4     per  of  the  sporangium.     Drawn  from  living  material. 

,        .                                                         .        .       .  E,  columella  with  a  few  spores  adhering.     F-H, 

cent.]      m      50      per      cent,     alcohol,  stages  in  the  formation  of  the  zygospore.     From 

a    preparation     fixed     in    corrosive    sublimate, 

used     hot,     may    be    recommended  stained  in  Delafleld's  hsematoxylln,  and  mounted 

•'  in  glycerine. 

as  a  fixing  agent.     Haem-alum,  or 

Delafield's  haematoxylin,  is  good  for  glycerine  preparations. 
Do  not  stain  too  deeply.  A  very  satisfactory  study  may  be 
made  from  the  living  material. 

Cystopus. — This  fungus  is  quite  common  on  Cruciferae,  where 
the  white  "blisters"  or  "white  rust"  form  quite  conspicuous 
patches. 

Affected  portions  of  leaves  and  stems  should  be  fixed  in 
chromo-acetic  acid  and  cut  in  parafifin.  Sections  5  /*  or  less  in 
thickness  will  be  found  most  satisfactory.  Safranin-gentian 
violet-orange  seems  to  be  the  best  stain  for  differentiating  the 
nuclei.      {Fig.  33.) 


Fig.  32.     Mucor  stolonifer.     X  255. 


8o  Methods  i?i  Plant  Histology 

It  is  more  difficult  to  get  good  sec- 
tions of  the  plant  in  the  oosporic  condi- 
tion. The  oosporic  phase  of  Cystopus  bliti 
is  easily  recognized  on  Antarantus,  where 
the  oospores  may  be  seen  with  the  naked 
eye  by  holding  the  leaf  up  to  the  light. 
While  better  nuclear  staining  can  be  se- 
cured with  chromic  or  Flemming  mate- 
rial, it  will  be  found  somewhat  easier  to 
cut  material  which  has  been  fixed  in  pic- 
ric acid  (i  per  cent,  solution  in  70  per 
cent,  alcohol).  Celloidin  sections,  stained 
in  Delafield's  haematoxylin,  can  be  rec- 
"^^^oT^Tpf^^c  ommended    for    showing    the    position    of 

Fig.  33.     Cystopus  candidus         ..  .  ,  ,  i         •   i  •  1,1  1  1 

on  Capseiia.  oogonia  and  anthendia,  although  such  sec- 

leaT^Ti^s'Ftma'X-atlon'  ^ions  are  too  thick  to  givc  satisfactory  views 

fixed  in  Flemming's  fluid  and  f  i.V-„  nnrlpi 
stained  in  safranin-gentian  vio-  *-"■  '■**'-  iiui-ici. 
let-orange. 

ASCOMYCETES. 

This  group,  popularly  known  as  the  "sac  fungi,"  contains  an 
immense  number  of  saprophytic  and  parasitic  forms.  Yeast, 
green  mold  on  cheese  and  leather,  leaf  curl  of  peach,  black 
knot  of  cherry  and  plum,  and  the  powdery  mildews  are  familiar 
to  everyone.  The  few  objects  selected  will  enable  the  student 
to  experiment,  but  he  must  not  be  discouraged  if  success  does 
not  crown  the  first  attempt,  for  the  group  presents  many  diffi- 
culties. 

Saccharomyces. —  Until  somewhat  recently  it  was  considered 
rather  difficult  to  demonstrate  the  nucleus  of  the  yeast  cell. 
With  fresh  growing  yeast  the  following  method  by  Wager 
should  be  successful :  Fix  in  a  saturated  aqueous  solution  of 
corrosive  sublimate  for  at  least  twelve  hours.  Wash  success- 
ively in  water,  30  per  cent,  alcohol,  70  per  cent,  alcohol,  and 
methyl  alcohol.  Place  a  few  drops  of  alcohol  containing  the 
cells  on  a  cover,  and  when  nearly  dry  add  a  drop  of  water. 
After  the  yeast  cells  settle,  drain  off  the  water  and  allow  the 


Thallophytes.     Fufigi 


8i 


cells  to  dry  up  completely.  Place  the  cover,  or  slide,  with  its 
layer  of  cells  in  water  for  a  few  seconds,  and  then  stain  with  a 
mixture  of  fuchsin  and  methyl  green,  or  fuchsin  and  methylen 
blue.     Mount  in  glycerine  or  in  balsam. 

Eurotium. —  For  class  use  or  for  permanent  preparations  it  is 
best    to    select 
rather    y  oung£^f^. 
material    which  *'"'' 
shows    various 
stages   in   devel- 
opment, from  the 
swollenendof  the 
hypha  to  the  ripe 
spore    [fig.  34) . 


C  D 

F;g.  34.     Eurotium. 


From   material  growing  on   a  hectograph  pad.     Fixed  in  chromo-acetic 
,  acid,  stained  in  eosin,  and  mounted  in  glycerine,     ^-^'j  successive  stages  in 

The    nuclei    are   development,   x  375- 

exceedingly  small,  and   can  hardly  be  demonstrated  with  eosin. 

Iron  alum-haematoxylin  would  be  better. 

The  following  schedule  will  give  good  mounts  for  habit  study: 

1.  Fix  in  a  saturated  solution  of  corrosive  sublimate  in  50  per  cent,  alcohol. 
The  addition  of  i  cc.  of  glacial  acetic  acid  to  100  cc.  of  this  solution 
improves  it.     Use  it  hot. 

2.  After  it  cools,  transfer  to  50  per  cent,  alcohol  and  add,  a  few  drops  at  a 
time,  the  iodine  solution  which  is  used  in  testing  for  starch.  At  first  the 
brownish  color  caused  by  the  iodine  will  disappear,  but  after  a  certain 
amount  has  been  added  the  brown  color  will  remain.  The  material  is 
then  ready  for  the  next  step. 

3.  Thirty-five  per  cent,  alcohol,  5  minutes. 

4.  Eosin  (aqueous),  at  least  5  minutes ;  a  day  will  do  no  harm. 

5.  Put  the  material  into  i  per  cent,  acetic  acid  for  15  seconds  to  2  minutes. 
The  material  should  still  have  a  vivid  red  color  when  taken  from  the  acid. 

6.  Wash  in  water.  Use  a  considerable  volume  of  water  or  change  the  water 
several  times.    If  the  acid  is  not  all  washed  out,  the  preparations  will  fade. 

7.  Ten  per  cent,  glycerine,  and  allow  the  glycerine  to  thicken. 

8.  Mount  in  glycerine  or  glycerine  jelly,  and  seal. 

Any  of  the  filamentous  fungi — like  Mucor,  Thamnidiutn, 
Peronospora,  Penicillium,  Pythium,  Saprolegnia,  etc.  —  can  be 
mounted  in  this  way.  Saprolegnia,  however,  is  much  more  satis- 
factory if  stained  in  iron  alum-haematoxylin. 


82 


Methods  in  Plant  Histology 


A  very  rapid  method  for  the  unicellular  and  filamentous 
forms  may  be  added  : 

1.  One  hundred  per  cent,  alcohol,  2  minutes. 

2.  Eosin  (aqueous),  2  minutes. 

3.  One  per  cent,  acetic  acid,  2  to  10  seconds. 

4.  Wash  in  water  5  minutes,  changing  frequently. 

5.  Mount  directly  in  50  per  cent,  glycerine,  and  seal. 

If  the  material  gets  through  the  first  four  stages  without 
shrinking,  but   collapses   at   the  fifth,  put   it  into    10  per  cent. 

glycerine  and  allow  it  to 
thicken  as  usual. 

Uncinula  necator. — 
The  mildews  are  found 
throughout  the  summer 
and  autumn  on  the  leaves 
of  various  plants.  The 
lilacmildew(  MicrosphcBra 
alni^  and  the  mildew  on 
the  Virginia  creeper 
{^Uncinula  necator)  are 
particularly  abundant. 
For  herbarium  purposes 
they  may  be  preserved 
by  simply  drying  the 
leaves  under  light  pres- 
sure. When  mounted  for 
examination,    the    leaf 

Fig.  35.    yl,  Uncinula  necator  on  Ampelopsis  quinquefolia.  should  bc  SOakcd  in  WatCr 

X  192.     Four  asci  containing  ascospores  have  been  forced  out  -  .  •        l  Ct. 

by  pressing  out   the  cover.     Fixed  in  hot  corrosive  sublimate,  lOr    3.    iCW     mmUtCS,   alter 
stained  in  fuchsin,  and  mounted  in  balsam.     /?,  a  conidiospore ;         ,    .    ,       . 

and  C,  an  appendage  of  Microsphaera  alni,  drawn  from  living  whlch  the  perithCCia  may 
material.     X  192.  '■  •' 

be  scraped  off  and 
mounted  in  water.  The  asci  may  then  be  forced  out  by  press- 
ing smartly  on  the  cover.      {Fig.  35.) 

For  permanent  mounts  of  entire  perithecia  with  appendages, 
fix  in  3  per  cent,  formalin  twenty-four  hours,  wash  in  water  one 
hour,  stain  in  aqueous  eosin  twenty-four  hours,  treat  with  i  per 


JOUR  APR  MIC. 


Thallophytes.     Fungi 


83 


cent,  acetic  acid  one  minute,  wash  thoroughly  in  water,  and  then 
transfer  to  10  per  cent,  glycerine,  which  should  be  allowed  to 
concentrate  as  usual.  If  chromic  acid,  corrosive  sublimate,  or 
alcohol  be  used  for  fixing,  the  appendages  become  brittle,  and 
very  easily  break  off.  However,  the  chromo-acetic  mixtures 
are  better  if  it  is  desired  to  make  parafifin  sections  showing  the 
development  of  the  perithecium  with  its  asci  and  spores.  For 
this  purpose  the  omnipresent 
Erysiphe  commune  on  Polygonum 
aviculare  is  exceptionally  favor- 
able, because,  after  the  material 
is  fixed  and  in  alcohol,  the  whole 
mycelium,  with  the  developing 
perithecia,  may  be  stripped  from 
the  leaf  without  the  slightest 
difficulty,  thus  avoiding  the 
necessity  of  cutting  the  leaf  in 
order  to  get  the  fungus.  The 
safranin- gentian  violet -orange 
combination  seems  to  give  the 
best  results,  although  cyanin 
and  erythrosin  are  quite  satis- 
factory when  the 
properly  balanced. 

■V—l^-i-  TV/r  p  i-1  several  days   in  equal  parts  of  gS  per  cent,  alcohol 

Aylaria. Many  forms,  like     and  glycerine,  and  then  imbedded  in  celloidin.    Not 

Xylaria,  Ustilina,  Hypoxylon,  and 

Nummularia,  in  their  mature  condition,  are  woody  and  so 
extremely  brittle  that  it  is  almost  impossible  to  cut  them.  As 
good  a  plan  as  any  seems  to  be  to  cut  sections  of  the  stroma 
about  one-eighth  of  an  inch  thick,  soak  them  in  equal  parts  of 
glycerine  and  95  per  cent,  alcohol,  and  then  imbed  them  in 
celloidin  in  the  usual  way.  They  might  be  cut  without  imbed- 
ding, but  most  of  the  asci  and  spores  would  then  be  lost. 

The  younger  stages,  showing  the  development  of  perithecia 
and  asci,  are  more  interesting,  and  can  be  cut  in  paraffin  and 
stained  with  ease  {fig- 36). 


A ,  transverse  section  of  a  young  stroma  showing 
stains      are     PS"*hecia.      X  8.       Fixed    in    chromo-acetic    acid, 
stained  in  bulk  in  alum  carmine,  imbedded  in  celloi- 
din, and    mounted    in   balsam.      B,   two  asci   with 
spores.     X  245.     The  mature  stroma  was  soaked  for 


Methods  i?i  Plant  Histology 


Peziza. —  The  Pezizas  and  related  forms  are  fleshy,  and  pre- 
sent but  little  difficulty  in  fixing,  cutting,  or  staining.  They  are 
abundant  in  moist  places,  on  decaying  wood, 
or  on  the  ground.  The  apothecia  have  the 
form  of  little  cups,  which  are  sometimes 
black  and  sometimes  flesh -colored,  but 
often  orange,  red,  or  green.  For  the  devel- 
opment of  ascospores  in  the  ascus,  Flem- 
ming's  fluid  (weaker  solution),  followed  by 
safranin-gentian  violet-orange,  has  given  the 
best  results  with  thin  sections  where  the 
mitotic  figures  are  to  be  studied.  Cyanin 
and  erythrosin  is  also  to  be  recommended. 
Such  sections  should  not  be-  more  than 
5  /*  in  thickness.  For  a  general  morpho- 
logical preparation,  such  as  is  shown  in 
the  figure,  it  is  better  to  stain  in  bulk  in 
alum  carmine  or  in  Delafield's  haematoxy- 
lin,  and  then  tease  out  the  asci  in  gly- 
cerine or  balsam.  Sections  thick  enough 
to  show  the  entire  ascus  are  not  usually 
as  satisfactory  as  such  teased  preparations. 
{Fig- 37-) 


Fig.  37.  Peziza  odorata. 
Three  asci  and  many  para- 
physes.  X  245.  Fixed  in  cor- 
rosive sublimate,  stained  in 
bulk  in  alum  carmine.  Teased 
out  and  mounted  in  balsam. 


^CIDIOMYCETES. 


The  .^cidiomycetes  comprise  the  rusts  [Uredine<^)  and  the 
smuts  [UstilaginecB). 

Puccinia  graminis. — The  common  rusts  of  wheat  and  oats 
are  familiar  to  everyone.  The  uredospores,  or  summer  spores, 
known  as  the  red  rust,  and  the  teleutospores  (last  spores),  or 
winter  spores,  known  as  the  black  rust,  are  found  in  unfortunate 
adundance,  but  the  aecidium  stage  on  the  barberry  is  not  neces- 
sary for  the  vigorous  development  of  rust  in  the  United  States, 
and  is  seldom  found.  Most  teachers  are  obliged  to  depend  upon 
botanical  supply  companies  for  this  material.  There  are,  how- 
ever,  various   aecidia  which   are    as    good,  or   even    better,   for 


Thallophytes.     Fungi 


&5 


morphological  study.  The  aecidia  growing  on  Euphorbia  maculata 
(spotted  spurge),  and  on  AriscEma  triphy Hum  (Jack-in-the-pulpit) 
are  much  easier  to  cut,  and  seem  easier  to  stain.  Delafield's 
haematoxylin,  followed   by  a  very   light   stain   in   erythrosin,  is 


tl/OURAPBMie.. 

Fig.  38.     Puccinia  graminis. 

_A,  transverse  section  of  barberry  leaf  showing  secidia  and  spermagonia.  X  7.  B,  longitudinal 
section  of  a  single  aecidium.  X  192.  Fixed  in  Flemming's  weaker  solution  and  stained  in  Delafield's 
haematoxylin.  C,  a  single  sperraagonium.  X  192.  Fixed  and  stained  as  in  B.  D,  three  uredospores 
growing  on  oats.  X  375.  Fixed  in  2  per  cent,  formalin,  stained  in  bulk  in  alum  carmine,  and  teased 
out  in  glycerine.  E,  section  of  young  teleutospores  on  oats.  X  375.  Fixed  in  picro-acetic  acid  and 
stained  in  cyanin  and  erythrosin.  G,  F,  //,  three  ripe  teleutospores  from  a  leaf  of  oats  showing  varia- 
tion in  form.     X  375.     /.  germinating  teleutospores.     X  375. 


II  ^  II 


Fig.  39. 


86  Methods  in  Plant  Histology 

good  for  both  aecidia  and  spermagonia,  especially  after  Flem- 
ming's  fluid.  It  is  rather  difficult  to  get  good  sections  of  uredo- 
spores  and  teleutospores,  because  the  leaves  of  wheat  and  oats 
are  refractory  objects  to  cut.  The  cutting  is  easier  after  picro- 
acetic  acid  than  after  corrosive  sublimate  or  the  chromic-acid 
series.      {Fig- jS.) 

Every  class  which  studies  the  rusts  should  attempt  to  germi- 
nate the  uredospores  and  teleutospores.  For  this  purpose  the 
hanging  drop  culture  may  be  employed.  Cement  a  rubber  or 
zinc  ring  to  the  slide,  or  simply  smear  the  lower  surface  of  the 
ring  with  vaseline  and  press  it  tightly  against  the  slide ;  smear 

the  upper  surface  of  the  ring 
with  vaseline,  and  over  it  invert 
ID  the  cover-glass  with  a  shallow 
drop  of  water  containing  the 
spores  {fig-  39).  The  uredo- 
spores germinate  readily  all  summer,  but  it  is  said  that  the 
teleutospores  will  germinate  only  in  the  spring  following  their 
maturity.  However,  the  teleutospores  of  many  species,  like 
Puccinia  xanthii  on  Xaftthium  canaderise  (cocklebur),  will  germi- 
nate as  soon  as  they  ripen  and  will  serve  equally  well  for  study. 
If  a  particularly  good  specimen  is  secured,  it  may  be  preserved 
by  the  method  previously  described  for  desmids,  except  that  in 
this  case  it  might  be  worth  while  to  attempt  staining  with  Mayer's 
haem-alum,  or  with  eosin. 

The  smuts  may  be  studied  in  the  living  material.  The  fol- 
lowing method,  recently  described  by  Ellis,  is  worth  remember- 
ing :  A  supply  of  smutted  barley  may  be  obtained  by  sowing 
soaked,  skinned  barley  that  has  been  plentifully  covered  by 
Ustilago  spores.  In  such  material  it  is  easy  to  trace  stages  in 
the  development  of  spores.  Free-hand  sections  of  ears  about 
three-eighths  of  an  inch  long  show  the  mycelium  and  spore  clus- 
ters. If  smutted  ears  be  removed  and  kept  floating  on  the 
water,  the  spores  continue  to  develop  and  often  germinate.  For 
paraffin  sections  desirable  stages  should  be  fixed  in  Flemming's 
fluid  or  picro-acetic  acid.     Delafield's  haematoxylin,  followed  by 


Thallophytes.     Fungi  87 

a  very  light  touch  of  erythrosin  or  acid  fuchsin,  will  give  a  good 
stain. 

For  a  study  of  the  germinating  spores  and  conidia,  cultures 
may  be  made   in  beerwort  on  the  slide  or  in  watch  crystals. 
Harper's  method  of  making  preparations  from  such  material  is 
ingenious  and  will  undoubtedly  prove  valuable  in  making  mounts 
of  various  small  plant  and  animal  forms,    A  drop  of  the  material 
is  taken  up  with  a  capillary  tube  and  is  then 
gently  blown  out  into  a  drop  of   Flemming's 
weaker  solution  (fifteen  minutes  or  an  hour  was 
sufficient    for    the    fungus    spores).     Cover    a 
slide  with  albumen  fixative,  as  if  for  sections. 
A  drop  of  the  material,  without  previous  wash- 
ing, is  drawn  up  into  the  capillary  tube  and 
touched  lightly  and  quickly  to  the  surface  of 
the  albumen.     A  series  of  such  drops,  almost 
as  small  as  the  stippled  dots  in  a  drawing,  may 
be  applied  to  the  slide.     The  fixing  agent  may 

Transverse  section  of    a 

now   be  allowed   to  evaporate  somewhat,  but  portion  of  one  of  the  giiis 

*  snowing  a  part  of  the  trama, 

the  preparation  must  not  be  allowed  to  dry.  ''•  and  several  basidia  ,j, 

r       r  J        each  with  four  sterigmata, 

As   the   slide    is    passed    rapidly  through  the  t^^^^ly^TAf^Jl 
alcohols,  the  albumen  is  coagulated,  and  the  ™*'''  ^"°* 
preparation  may  be  treated    just  as  if  one  were  dealing  with 
ribbons  of  sections. 

BASIDIOMYCETES. 

This  is  an  immense  group,  of  which  the  mushrooms,  toad- 
stools, puffballs,  and  bracket  fungi  are  the  most  widely  known 
representatives. 

Coprinus  comatus. — This  is  the  common  shaggy-mane  mush- 
room. Cut  from  the  cap  pieces  about  one-fourth  of  an  inch 
square,  and  fix  in  chromo-acetic  acid  or  in  Flemming's  fluid.  Por- 
tions in  which  the  gills  have  just  begun  to  turn  brown  will  show  the 
spores  still  attached  to  the  sterigmata  {fig.  40) .  If  the  gills  have 
become  dark  brown  or  black,  the  spores  will  wash  off  before  the 
sections  can  be  mounted.  Look  in  portions  in  which  the  gills 
are  still  white  or  only  slightly  changing  color  for  the  develop- 


88  Methods  in  Pla?it  Histology 

ment  of  basidia  and  spores.  The  nuclei,  although  rather  small, 
are  brought  out  nicely  by  safranin-gentian  violet-orange.  The 
same  procedure  may  be  observed  for  other  forms  of  similar  con- 
sistency, like  many  members  of  the  genera  Boletus,  Hydnum, 
Polyporus,  Lycoperdon,  etc.  Leathery  or  woody  forms  like  Stereum 
and  many  species  of  Polyporus  had  better  be  fixed  in  picro-acetic 
acid  and  imbedded  in  celloidin.  Young  stages  of  Cyathus  or 
Crucibulum  (bird's-nest  fungi)  cut  easily  in  paraffin,  but  the 
older  stages  cut  much  better  in  celloidin.  It  is  hard  to  get  the 
very  soft,  watery  forms  like  Tremella  into  paraffin  without  shrink- 
ing, but  sections  as  thin  as  lo  /u-  may  be  cut  in  celloidin.  While 
this  is  too  thick  to  give  satisfactory  views  of  such  small  nuclei, 
it  brings  out  very  clearly  the  general  morphological  structures. 

THE  LICHENS. 

The  lichens  are  usually  regarded  as  diflficult  forms.  In 
younger  stages  they  occasion  no  trouble,  but  an  old  apothecium 
or  a  leathery  thallus  often  fails  to  cut  well.  Difficulties  may  be 
minimized  by  using  prolonged  periods.  The  following  schedule 
has  proved  satisfactory  for  the  thalli  and  mature  apothecia  of 
Physcia,  Usnea,  Sticta,  Collema,  Parmelia,  and  Peltigera: 

1.  Chromo-acetic  acid  (medium  solution,  p.  28),  2  to  4  days. 

2.  Wash  in  water,  6  to  24  hours. 

3.  Thirty-five,  50,  70,  85,  and  95  per  cent,  alcohols,  6  to  24  hours  each. 

4.  One  hundred  per  cent,  alcohol,  2  to  4  days,  changing  2  or  3  times. 

5.  Mixtures  of  alcohol  and  xylol,  i  to  2  days. 

6.  Pure  xylol,  6  to  24  hours. 

7.  Xylol  and  parafifin  on  the  bath,  i  to  2  days. 

8.  Parafifin  at  54°  to  60",  changing  once  or  twice,  3  to  6  days. 

9.  Imbed  in  as  thin  cakes  as  possible. 

Cyanin  and  erythrosin  is  a  very  good  stain  for  lichens.  The 
algae  stain  blue  and  the  filaments  of  the  fungus  take  the  red. 
Where  the  association  of  the  alga  and  the  fungus  is  rather  loose, 
as  in  Dichonema,  more  satisfactory  mounts  can  be  made  by  stain- 
ing in  eosin,  or  haem-alum  and  eosin,  and  then  teasing  slightl}' 
with  needles  and  mounting  in  glycerine. 


CHAPTER   XIII. 

BRYOPHYTES. 

The  Bryophytes,  comprising  the  two  groups  Liverworts 
[Hepaticcs)  and  Mosses  [Mtisci] ,  present  a  great  diversity  of 
structure,  some  being  so  delicate  that  good  preparations  are  very 
uncertain,  while  others  are  so  hard  that  it  is  difficult  to  get  satis- 
factory sections.  Between  these  extremes,  however,  there  are 
many  forms  which  readily  yield  beautiful  and  instructive  prepa- 
rations. 

If  but  one  fixing  agent  should  be  suggested  for  the  entire 
group,  it  would  be  chromo-acetic  acid  with  ^  g.  chromic  acid 
and  ^  cc.  acetic  acid  to  lOO  cc.  of  water.  -  It  should  be  allowed 
to  act  for  at  least  twenty-four  hours,  and  probably  two  or  three 
days  would  be  better.  Always  make  an  effort  to  get  the  mate- 
rial into  paraffin,  using  celloidin  only  as  a  last  resort  for  refrac- 
tory structures  which  resist  infiltration  and  for  very  delicate 
structures  which  persist  in  collapsing.  As  one  gains  in  experi- 
ence and  carefulness,  the  number  of  cases  which  seem  to  demand 
celloidin  will  become  fewer  and  fewer. 

Instead  of  treating  forms  in  a  taxonomic  sequence,  we  shall 
consider  first  the  gametophyte  structures  under  the  headings 
thallus,  antheridia,  and  archego?iia,  and  shall  then  turn  our  atten- 
tion to  the  sporophyte. 

HEPATIC-S:. 

Some  of  the  liverworts  are  floating  aquatics,  but  most  of 
them  grow  on  logs  or  rocks  or  upon  damp  ground.  They  are 
found  at  their  best  in  damp,  shady  places.  Many  of  them  may 
be  kept  indefinitely  in  the  greenhouse.  Riccia,  Ricciocarpus, 
Marcha?itia,  Cofiocephalus,  Asterella,  and  many  others  vegetate 
luxuriously,  and  often  fruit  if  kept  on  moist  soil  in  a  shady  part 
of  the  greenhouse,  and  they  do  fairly  well  in  the  ordinary  labo- 
ratory if  covered  with  glass    and  protected   from   too   intense 

89 


90 


Methods  in  Plant  Histology 


Fig.  41 
A,  longitudinal. 


Ptilidium  ciliare.     X  420. 
8,  transverse  section  of  the  apex  of  the 


leafy  gametophyte.     Fixed  in  Flemming's  weaker  solution, 
stained  in  a  mixture  of  acid  fuchsin  and  iodine  green.    Ten 


light.  The  living  plants  are  very  desirable,  since  they  not  only 
furnish  the  best  possible  material  for  habit  work  and  the  coarser 
microscopic  study,  but  they  also  enable  one  to  secure  complete 
series  in  the  development  of  the  various  organs. 

The  Thallus. —  In  many  cases  it  will  not  be  necessary  to  make 
a  special  preparation  for  the  study  of  the  thallus,  since  prepara- 
tions of  antheridia,  archegonia,  or  sporophytes  may  include  good 

sections  of  vegetative  por- 
tions. This  is  particularly 
true  of  forms  like  Riccia, 
where  the  various  organs 
are  not  raised  above  the 
thallus.  In  forms  like  J/<2r- 
chantia,  where  the  antheri- 
dia, archegonia,  and  sporo- 
phytes  are  borne  upon 
stalked  receptacles,  it  is 
better  to  make  separate  preparations  to  show  the  structure  of 
the  mature  thallus.  Sections  intended  to  show  the  structure 
of  the  mature  thallus  should  be  15  ft  to  25  /x  in  thickness,  but 
sections  to  show  the  growing  point  and  development  of  the 
thallus  should  not  be  thicker  than  10  /u..  Material  showing  apical 
cells  and  development  of  the  thallus  is  easily  gotten  into  paraf- 
fin, even  in  forms  like  RicciocarpuSy  which  in  their  mature  condi- 
tion are  in  danger  of  collapsing.  The  apical  region  of  the  foliose 
Jungermanniacecs  {fig.  41)  affords  an  excellent  opportunity  for 
studying  the  development  of  the  plant  body  from  a  single  apical 
cell.  If  mixtures  containing  osmic  acid  are  used  for  fixing, 
there  may  be  difficulty  in  the  staining,  even  after  using  per- 
oxide of  hydrogen.  Corrosive  sublimate-acetic,  Carnoy's  fluid, 
or  chromo-acetic  acid  are  better  for  apical  regions.  A  fairly 
vigorous  staiiiing  with  a  mixture  of  acid  fuchsin  and  iodine 
green  often  brings  the  walls  out  very  sharply.  Chromo-acetic 
acid,  followed  by  Delafield's  haematoxylin  or  bismark  brown,  is 
good  for  the  apical  cells  and  developing  regions,  but  a  light 
counter-stain    with    erythrosin    improves     preparations    of    the 


Bryophytes 


mature  thallus.  After  corrosive  sublimate-acetic  the  material 
maybe  stained  in  bulk  with  alum  cochineal  or  alum  carmine, 
thus  giving  fairly  good  preparations  and  saving  considerable 
labor. 

Antheridia. —  If  you  have  the  material,  it  is  not  difficult  to 
get  good  preparations  showing  the  development  of  antheridia. 
In  forms  like  Asterella,  Pellia,  etc.,  cut  out  a  small  portion  of  the 
thallus  bearing  the  antheri- 
dia. The  piece  should  not 
be  more  than  a  quarter  of 
an  inch  square,  and  if  it  can 
be  smaller,  so  much  the 
better.  For  early  stages  of 
the  antheridia  of  Marchan- 
tia  select  young  antheridio- 
phores  which  still  lie  close 
to  the  thallus.  These 
readily  cut  as  thin  as  5  /*, 
and  a  single  slide  will  usu- 
ally show  a  more  complete 
series  than  is  represented  in  the  figure  of  Asterella  {fi^.  42),  but 
after  the  stalk  begins  to  lengthen,  the  younger  stages  become 
infrequent,  and  it  is  not  always  easy  to  cut  thin  sections.  Dela- 
field's  haematoxylin  or  bismark  brown  serves  very  well  for  such 
stages  as  are  shown  in  the  figure.  The  protoplasm  of  the  young 
antheridia  is  so  dense  that  the  addition  of  a  counter-stain  is 
almost  sure  to  injure  the  preparation  by  obscuring  the  cell  walls. 
For  stages  older  than  that  represented  in  D,  showing  the  devel- 
opment of  the  spermatozoid,  the  paraffin  must  be  rather  hard 
(melting  at  55°  C.  to  65°  C.) ,  and  the  sections  should  not  be 
thicker  than  5  /a,  while  2  /it  or  3  /*  is  best.  For  such  stages  use 
the  safranin- gentian  violet -orange  combination,  Haidenhain's 
iron  alum-haematoxylin  with  or  without  a  faint  trace  of  eryth- 
rosin  or  orange  G,  or  use  a  mixture  of  acid  fuchsin  and  methyl 
green.  Nothing  but  practice  and  patience  will  bring  success  in 
such  critical  work. 


Successive  stages  in  the  development  of  antheridia. 
Fixed  in  chromo-acetic,  stained  with  Delafield's  haema- 
toxylin.    Section  lo  microns  thick. 


92 


Methods  i?i  Plant  Histology 


If  antherozoids  are  found  escaping,  transfer  them  to  a  small 
drop  of  water  on  a  clean  slide,  invert  the  drop  o\'er  a  i  per  cent, 
solution  of  osmic  acid  for  two  or  three  minutes,  allow  the  drop 
to  dry  up,  pass  the  slide  through  the  flame  two  or  three  times, 
as  in  mounting   bacteria,  and  then   stain  sharply  in  acid  fuchsin. 

This  should  show  the 
general  form  of  the  an- 
therozoid,  and  will  usu- 
ally bring  out  the  cilia. 

The  Archegonia. — The 
methods  for  archegonia 
are  practically  the  same 
as  for  antheridia.  Too 
much  stress  cannot  be 
laid  upon  the  importance 
of  carefully  selecting  the 
material.  Use  very  small 
pieces,  and,  before  pla- 
cing them  in  the  fixing 
agent,  trim  them  to  such 
a  shape  that  the  position 
of  the  archegonia  will  be 
accurately  known  even 
after  the  pieces  are  imbedded  in  paraffin.  For  stages  like^^.  4J, 
A  and  B,  Delafield's  haematoxylin  is  a  good  stain,  and  lo  /u  is 
about  the  right  thickness.  For  stages  like  C,  in  such  forms  as 
Marchantia,  where  the  necks  are  long  and  often  somewhat  curved, 
it  is  better  for  general  purposes  to  use  sections  from  1 5  yit  to 
20  [i  in  thickness.  If  it  is  desired  to  obtain  preparations  show- 
ing the  cutting  off  of  the  ventral  canal  cell,  the  development  of 
the  oosphere,  and  the  process  of  fertilization,  the  sections  should 
be  from  5  /i  to  10  /*  in  thickness,  and  the  same  staining  may  be 
used  as  for  the  development  of  antherozoids.  For  archegonia 
containing  young  embryos,  like  that  shown  in  D,  Delafield's 
haematoxylin  without  any  counter-stain  gives  beautiful  prepara- 
tions when  the  staining  is  well  done.     It  is  easier  for  the  beginner 


Fig.  43. 


Marchantia  polymorpha.     X  400. 


A,  three  early  stages  in  the  development  of  the  archegonia. 
Delafield's  haematoxylin.  B,  young  archegonium  showing  two- 
neck  canal  cells  and  the  central  cell  before  the  cutting  off  of 
the  ventral  canal  cell.  Fuchsin  and  methyl  green.  C,  mature 
archegonium  just  ready  for  fertilization.  Safranin-gentian 
violet-orange.     D,  young  embryo.      Delafield's  haematoxylin. 


Bryophytes 


93 


to  get  good  preparations  with  the  safranin-gentian  violet-orange 
combination. 

The  Sporophyte. —  Sporophytes  in  early  stages  of  develop- 
ment often  yield  good  preparations  without  very  much  trouble, 
but  in  later  stages  they  are  frequently  difficult  to  cut  on  account 
of  the  secondary  thickening  of  the  capsule 
wall  and  the  stubborn  extine  of  the  mature 
spores.  It  is  hard  to  get  Ricciocarpus  into 
paraffin  without  shrinking,  and  the  same 
thing  may  be  said  of  other  forms  which 
have  such  loose  tissue  with  large  air  cavi- 
ties. For  stages  like  that  shown  in  fig.  44, 
as  well  as  for  older  sporophytes,  it  will  be 
found  more  uniformly  satisfactory  to  use 
celloidin,  and  cut  the  sections  from  20  /x 
to  30  /A  thick. 

For  a  study  of  archegonia,  antheridia, 
young  sporophytes,  and  also  for  the  devel- 
opment of  the  thallus,  it  is  better,  even  in 


Ricciocarpus  natans. 


ig  sporophyte  inclosed  in 
the  archegonium.  Spore  mother- 
cell  stage.  All  the  cells  of  the 
sporophyte  except  a  single  peri- 
pheral layer  (dotted  in  the  figure) 
produce  spores.  Fixed  in  picro- 
acetic  acid  and  stained  in  Dela- 
field's  haematoxylin.  Celloidin 
section  30  microns  in  thickness. 

forms  like  Ricciocarpus, 
to  use  paraffin.  Pro- 
longed fixing  in  chro- 
mo-acetic  acid  (two  to 
six  days),  thorough 
dehydrating,  and 
gradual  transfer  from 
alcohol  to  xylol,  and 
from  xylol  to  paraffin, 
and  also  a  moderate  temperature  in  the  bath  (not  more  than 
50"  C),  will  often  bring  the  material  through  in  fine  condi- 
tion. 


Fig.  45.     Pellia  epiphylla. 

A,  habit  sketch  of  sporophyte.  X  10.  B,  small  portion  of 
sporophyte  (at  X  of  ^),  showing  the  capsule  wall,  the  spores, and 
the  elaters.  Fixed  in  chromo-acetic  acid  and  stained  in  cyanin 
and  erythrosin.     Ten  microns. 


94 


Methods  in  Plant  Histology 


Forms  like  Pellia  cut  well  in  paraffin,  especially  in  younger 
stages,  but  even  in  case  of  mature  sporophytes  it  is  not  neces- 
sary to  resort  to  celloidin.  In  Pellia  and  Conocephalus  the  spores 
are  very  large  and  have  a  rather  thin  wall.  Both  these  genera 
show  a  peculiar,  intrasporal  development  of  the  gametophyte, 
/.  e.,  the  gametophyte  develops  to  a  considerable  extent  before 
it  ruptures  the  spore  wall.  (  Fig.  ^5.)  For  the  older  sporophytes 
of  Marchantia  it  is  better  not  to  cut  the  whole  receptacle,  but 


Jou(i./|pp,A|it 


Fig.  46.    Anthoceros  laevis. 


A,  longitudinal  section  of  lower  portion  of  sporophyte  imbedded  in  the  gametophyte.  X  45.  B, 
transverse  section  of  lower  portion  of  sporophyte.  X  200.  Delafield's  hsematoxylin.  Ten  microns. 
C,  vegetative  cell  from  lower  portion  of  the  sporophyte.  X  560.  Fixed  in  Flemming's  weaker  solution 
and  stained  in  a  mixture  of  acid  fuchsin  and  iodine  green.  Five  microns.  D,  spore  mother-cell  showing 
three  of  the  four  chloroplasts  with  numerous  starch  grains.  The  nucleus  is  in  the  metaphase  of  the  first 
division.  X  560.  Fixed  in  Flemming's  weaker  solution,  stained  in  safranin-gentian  violet-orange. 
Five  microns. 

rather  to  remove  the  branches  so  that  they  may  be  cut  separately. 
For  the  very  best  preparations  of  mature  sporophytes  it  will  pay 
to  trim  away  the  gametophyte  structures,  leaving  only  enough  to 
show  the  foot  with  a  few  of  the  surrounding  cells.  Sections  5  /a 
to  10  /i  thick  can  be  made  without  much  difficulty  from  material 
prepared  in  this  way. 

Among   the    Bryophytes   no   form   affords   a    better   oppor- 
tunity for  studying  the  development  of  spores  than  Anthoceros, 


Bryophytes  95 

since  a  single  longitudinal  section  of  the  sporophyte  may  show- 
all  stages,  from  earliest  archesporium  to  mature  spores  {^fig.  46) . 
For  studies  like  A  and  B,  chromo-acetic  material  cut  10  /u,  thick 
and  stained  in  Delafield's  haematoxylin  is  very  good.  The 
starch  grains  in  the  chloroplasts  take  a  beautiful  violet  color 
with  the  safranin-gentian  violet-orange  combination.  It  is  very 
difficult,  however,  to  bring  out  the  details  of  nucleus  or  chloro- 
plast  on  account  of  the  minute  size  of  these  structures.  The 
drawings  from  which  C  and  D  were  reproduced  were  made  with 
a  one-sixteenth  oil  immersion  objective.  The  drawings,  like  all 
the  others  illustrating  the  Bryophytes,  were  reduced  one-half  by 
photography. 


CHAPTER  XIV. 


BRYOPHYTES. 

MUSCI. 

Material  for  a  study  of  the  mosses  is  much  more  abundant, 
and  a  series  of  stages  in  the  development  of  the  various  organs 
is  easily  secured ;  but  it  is  much  more  difficult  to  obtain  good 
preparations,  because  so  many  of  the 
structures  are  hard  to  cut.  Chromo- 
acetic  acid  is  to  be  recommended  as 
the  most  satisfactory  fixing  agent,  but 
where  structures  are  refractory  and  very 
likely  to  make  trouble  in  cutting,  it  will 
often  be  found  more  satisfactory  to  use 
picro- acetic  acid  in  the  70  per  cent, 
alcohol,  since  material  fixed  in  this 
reagent  does  not  become  as  hard  or  as 
brittle  as  that  fixed  in  any  of  the  chro- 
mic-acid series. 

Antheridia. —  It  is  easy  to  find  mate- 
rial for  a  study  of  antheridia,  because, 


Fig.  47- 


A,  archegonia  of  Webbera  candi- 
,1  ,1         •!•     1         1         .        cans.       X   104.       Celloidin     section. 

m  SO  many  cases,  the  anthendial  plants  Twenty  microns.    5,  young  amheri- 
can  be  detected  at  once  without  even  a 


Polytrichum 


.    X420. 


pocket  lens.  Funaria,  with  its  bunch  of  antheridia  as  large  as  a 
pin-head,  is  extremely  common  everywhere.  Spring  is  the  best 
time  to  collect  it,  but  it  is  found  fruiting  in  the  autumn  and 
sometimes  in  summer ;  besides,  it  is  easily  kept  in  the  green- 
house, where  it  may  fruit  at  any  time.  Bryum  proliferiim.  has  a 
still  larger  cluster  of  antheridia,  which  may  be  seen  at  a  distance 
of  several  yards.  Polytrichum  also  has  a  large  cluster  of  anthe- 
ridia surrounded  by  reddish  leaves,  so  that  the  whole  is  some- 
times called  the  moss  "flower."  In  making  preparations  of 
Polytrichum  these  colored   leaves  should   be  carefully  removed 

97 


98 


Methods  in  Plant  Histology 


after  the  material  has  been  gotten  into  70  per  cent,  alcohol.  A 
single  antheridial  plant  of  Polytrichum  often  furnishes  a  fairly 
complete  series  of  stages  in  the  development  of  antheridia. 
{Fig.  47.)  In  all  cases  the  stem  should  be  cut  off  close  up  to 
the  antheridia,  for  many  of  the  moss  stems  cut  like  wire.  It  is 
not  necessary  to  use  celloidin  for  antheridia,  nor  is  it  desirable, 

except  where  sec- 
tions from  20  /A  to 
50  /A  thick  are  want- 
ed for  habit  work. 
Delafield's  haema- 
toxylin  is  recom- 
mended for  staining. 
Archegonia. — 
Since  the  necks  of 
the  archegonia  are 
usually  long  and 
more  or  less  curved, 
it  is  necessary,  for 
habit  work,  to  cut 
sections  as  thick  as 
20  /i  or  30  ft  in  or- 
der to  get  a  view  of 
Jm.flw.A1iQ>  an  archegonium  in  a 
sinsfle  section.    Cel- 


FiG.  48.     Funaria  hygrometrica. 


endotheciu 
)elafield 
microns.    B,  C,  and  D,  transverse  sections 
age  as  .^,  taken  at  three  different  levels.     X  255.    Ten  microns. 


and  amphithe- 


A,3ipex  of  young  sporophyte  showing  < 

X  420.     Chromo-acetic  acid  and  Delafield's  haematoxylin.    Ten    loidin    mav   be    USCd 
of  a  sporophyte  of  the  same  -^ 


for  such  prepara 
tions,  but  for  the  development  of  the  archegonium,  the  oosphere, 
the  canal  cells,  and  also  for  the  process  of  fertilization,  it  is  better 
to  use  paraffin.  For  the  thick  celloidin  sections  the  material 
may  be  stained  in  bulk  in  alum  cochineal,  but  thin  paraffin  sec- 
tions should  be  stained  on  the  slide  with  more  critical  stains. 

{^^^-  47-) 

The  Sporophyte. —  It  is  often  difficult  to  get  good  mounts  of 
sporophytes.  In  the  younger  stages  the  calyptras  are  likely  to 
interfere  with  cutting,  while  in  the  older  stages  the  peristome,  or 


Bryophytes 


99 


hard  wall  of  the  capsule,  occasions  the  trouble.  If  an  attempt  is 
made  to  remove  the  calyptra  in  young  stages,  like  A  oi  fig.  48, 
the  apex  of  the  sporophyte  usually  comes  with  it.  While 
picro-acetic'  acid  material  cuts  more  easily,  chromo-acetic  acid 
followed  by  Delafield's  haematoxylin  gives  so  much  sharper  dif- 
ferentiation in  stages  like  those  shown  in  fig.  48  that  it  is  better 


hm^.Hii. 


Fig,  49.     Funaria  hygrometjica.     X  420. 

^.longitudinal  section  of  capsule.  B,  transverse  section  of  capsule  of  about  the  same  age  as^. 
The  columella,  archesporium,  outer  spore  case,  two  layers  of  chlorophyll-bearing  cells,  and  the  beginning 
of  the  air  spaces  can  be  distinguished  at  this  stage.  Delafield's  haematoxylin  and  erythrosin.  Ten 
microns. 

to  use  harder  paraffin  (55°  to  60°  C.)  and  make  an  effort  to  get 
preparations  from  chromic  material. 

Stages  like  that  shown  in  fig.  4g  are  cut  with  comparative 
ease,  for  the  calyptra  is  easily  removed,  and  the  capsule  wall  is 
not  yet  hard  enough  to  occasion  any  difficulty.  The  cell  walls 
are  so  easily  stained  in  moss  capsules  that  a  light  counter-stain 
with  erythrosin  or  acid  fuchsin  may  be  used  to  bring  out  the 


100 


Methods  in  Platii  Histology 


cytoplasm  and  plastids  without  appreciably  obscuring  the  cell 
walls.  Funaria  and  Bryum  afford  an  excellent  study  in  the 
development  of  the  capsule,  since  all  the  structures  of  a  highly 
differentiated  moss  sporophyte  are  present,  and  Bryum  is  par- 
ticularly easy  to  cut  in  stages  like  those 
shown  in  fig.  §o. 

Sporophytes,  in  their  more  mature 
stages,  are  almost  sure  to  present  con- 
siderable difficulty  in  cutting.  For  gen- 
eral work  fairly  good  preparations  may 
be  gotten  from  celloidin  material,  but  it 
is  worth  while  to  try  paraffin,  for  it  is 
sometimes  successful,  and  when  it  does 
succeed  -it  is  far 
Jow/j»;y,c  superior.    As  soon 

Fig.  so.     Bryum.     X  200.  aS     the     CcU     Walls 

Portion  of  a  nearly  mature  capsule  K^rrin     fr>     tViirl^^n 

showing  operculum,  annulus,   peris-  DCgm     tO     iniCKCn, 
tome,    and   three  cells  of  the   sporo-  •       .1^       rl<^ir/:»1r.r-i 

genous  tissue.     Fixed  in   Flemming's  ^S    m   tUC   aeVCiOp- 
weaker  solution,   stained   in   safranin  j.        £    tU 

and  Delafield's    haematoxylin.      Fif-  mCUt    Ot    the    pCri- 
teen  microns.  . 

stome,  safranm  is 
an  excellent  stain,  and  this,  followed  by 
Delafield's  haematoxylin,  will  give  an  ele- 
gant differentiation  in  the  older  stages  of 
the  sporophyte.  After  capsules  have  be- 
gun to  turn  brown  it  will  be  almost  impos- 
sible to  infiltrate  them  unless  they  are 
pricked  with  a  needle. 

The  mature  sporophytes  of  Sphagnum 
'[fig-  51)  are  exceptionally  hard  to  cut.     It  -^  -^^'n  ry  jo^mMtnm 

will  be  worth  while  to   prick  the  capsule       '^"  ^''     ^  ^gnum.      24. 

^  *  Longitudinal  section  of  mature 

with  a  needle  when   the   material    is  col-  sporophyte,  showing  also  the  upper 


lected.     This  wil 


portion  of  the  pseudopodium  and 
allnw  thp  fivincr  pcrpnt  'he  calyptra.  Chromo-acetic 
ailOW    me     nxmg    agent     ^^j^^     Delafield's     hematoxylin. 

to  penetrate  readily,  and  will  also  facilitate  ^""*^"-  Ten  microns. 
the  infiltration  of    paraffin  or  celloidin.     The    puncture  causes 
only  a  slight  damage,  and  need  not  reach  the  really  valuable 
portion  which  is  to  furnish  the  median  longitudinal  sections. 


Bryophytes  lOi 

Protonema  and  teased  mounts  of  antheridia  and  archegonia 
may  be  made  directly  in  50  per  cent,  glycerine  without  fixing  or 
staining.  While  this  method  is  often  recommended,  we  have 
found  it  better  to  use  10  per  cent,  glycerine  and  allow  it  to  con- 
centrate. Mounts  made  in  this  way  retain  their  green  color  for 
a  long  time. 


CHAPTER  XV. 

PTERIDOPHYTES. 

This  group,  including  the  Filicineae,  Equisetineae,  and  Lyco- 
podineae,  or,  more  popularly,  the  ferns,  horsetail  rushes,  and 
club  mosses,  is  familiar  to  everyone.  Material  is  abundant, 
and  so  easily  recognized  that  anyone  who  pays  a  little  attention 
to  collecting  can,  in  a  single  season,  get  a  fine  supply  for  a  study 
of  the  group.  Some  desirable  forms  may  not  be  present  in  all 
localities,  but  these  will  be  few  and  can  be  obtained  at  a  reason- 
able price  from  those  who  make  a  business  of  collecting. 

FILICINEiE. 

Without  attempting  to  follow  any  taxonomic  sequence,  the 
methods  of  preparing  the  various  structures  of  the  homosporous 
forms  will  be  presented,  and  then  the  peculiarities  of  the  hetero- 
sporous  members  will  be  considered. 

The  Prothallia. —  Ripe  spores  of  some  fern  or  other  can  be 
obtained  at  any  greenhouse  at  any  time  in  the  year,  and  spores 
of  most  of  our  native  ferns  germinate  well  and  produce  good 
prothallia,  even  if  the  sowing  is  not  made  for  several  months 
after  the  spores  have  been  gathered. 

Fine  prothallia  of  Pteris  aquilina  have  been  grown  two  years 
after  the  spores  were  gathered.  Some,  however,  must  be  sown 
at  once,  or  they  will  not  germinate  at  all.  The  spores  of  the 
common  Osmu?ida  regalis,  and  probably  of  the  other  members  of 
the  genus,  must  be  sown  as  soon  as  ripe,  or  they  fail  to  gefminate. 
The  prothallia  of  Osmu?ida  regalis,  if  carefully  covered  with 
glass,  may  be  kept  for  a  long  time.  Prothallia  of  this  fern  in 
the  writer's  laboratory  produced  ribbon-like  outgrowths  three- 
sixteenths  of  an  inch  wide,  and  often  more  than  two  inches  in 
length.  These  prothallia  continued  to  produce  archegonia, 
antheridia,  and  the  ribbon-like  outgrowths  for  more  than  a  year, 

103 


104  Methods  i?i  Plafii  Histology 

when  they  suddenly  "damped  off."  Pteris  aquilina  and  many 
other  ferns  often  furnish  a  good  supply  of  antheridia  in  three 
weeks  after  sowing,  and  the  archegonia  appear  soon  after,  but  it 
is  well  to  make  sowings  six  weeks  before  material  is  needed  for 
use.  In  Pteris  aquilina  and  in  many  others,  if  the  spores  are 
sown  too  thickly,  only  antheridial  plants  will  be  obtained.  If 
prothallia  are  to  produce  archegonia,  they  must  have  sufficient 
room  and  nutrition.  If  there  are  no  greenhouse  facilities  and 
the  prothallia  must  be  grown  in  the  laboratory,  it  is  a  good  plan 
to  take  a  glass  dish,  ten  or  twelve  inches  in  diameter  and  about 
two  inches  deep,  put  a  layer  of  broken  pieces  of  flower  pots  on 
the  bottom,  cover  this  with  a  layer  of  rich  loam,  and  over  this 
sprinkle  a  layer  of  fine,  clean  sand,  since  sand  is  much  more 
easily  washed  away  from  the  rhizoids  than  is  the  loam.  The 
whole  should  now  be  thoroughly  wet,  but  not  so  as  to  have 
water  standing  on  the  bottom.  Sow  the  spores  and  cover  with 
a  tightly  fitting  pane  of  ground  glass.  There  should  be  no 
need  for  moistening  the  culture  again,  for  prothallia  can  be  kept 
fresh  and  vigorous  for  several  months,  or  even  for  a  year,  without 
any  wetting.  When  it  is  desired  to  secure  fertilized  material, 
sprinkle  the  prothallia  with  water,  and  the  young  sporophytes 
will  soon  appear.  If  greenhouse  facilities  are  available,  any  gar- 
dener can  grow  prothallia  in  abundance  without  any  directions 
from  those  who  want  the  material. 

The  peculiar  tuberous  prothallia  of  Botrychium  are  seldom 
found  except  by  the  experienced  collector.  The  older  prothal- 
lia, however,  may  be  found  by  anyone  who  is  able  to  recognize 
Botrychium  when  he  sees  it.  Dig  up  young  plants  not  more 
than  three  or  four  inches  in  height,  and  the  prothallia,  which 
persist  for  years,  will  often  be  found  still  attached.  They  are 
easy  to  cut  and  may  be  handled  like  other  prothallia. 

Fern  prothallia  of  the  usual  type  are  excellent  objects  for 
testing  fixing  agents,  since  the  prothallia,  while  still  in  the  fixing 
agent,  may  be  examined  with  the  microscope,  and  fluids  which 
cause  plasmolysis  may  be  rejected.  It  will  sometimes  happen 
that  plasmolysis  may  be  avoided  by  varying  the  proportions  of 


Pteridophytes 


105 


the  ingredients  of  a  fixing  agent.  Chromo-acetic  acid  with 
about  0.6  g.  chromic  acid  and  0.4  cc.  of  acetic  acid  to  100  cc.  of 
water  will  seldom  cause  plasmolysis,  and  will  usually  insure  good 
fixing.  It  is  a  mistake  to  suppose  that  because  prothallia  are 
suchdelicateobjects 
the  fixing  will  take 
but  a  few  minutes. 
We  should  recom- 
mend at  least  twen- 
ty-four  hours  in 
chromo-acetic  o 
Flemming's  fluid 
and  two  or  three 
days  will  do  no  harm 
and  may  be  better. 
If  hot  corrosive  sub- 
limate-acetic acid  or 
hot  picro-acetic  be 
used,  the  fixing  re- 
quires only  two  or 
three  minutes,  but 
results  are  not  as 
uniformly  success- 
ful as  with  members 
of  the  chromic-acid 
series.      After    any 

Fig.  52.     Pteris  aquihua. 

of  the   ChromiC-aCld        ^,  filamentous  stage.     5,  the  apical   cell  has  been  established  and 

,  several   segments  have  been   cut  off.      The   figure  shovifs   the   initial 

series,    two    or    three     rhizold,  and   also   three   rhizoids   coming   from   the  main   body  of  the 

^  .         prothallium.     C,  an  older  prothallium  covered  with  antheridia  in  vari- 

hoUrS        washing       in     ous  stages   of  development.     From  a  glycerine  mount,  fixed  in  chromo- 

acetic   acid   and   stained   in   Deiafield's  hseraatoxylin.     (Miss  M.  E. 

water  will  be  sufifi-  tarrant.) 

cient,  if    the  water  be  changed  as  often  as  it  becomes  in  the 

least  degree  discolored. 

If  preparations  are  to  be  mounted  whole,  as  shown  in  fig.  j2, 
they  should  be  stained  as  soon  as  the  washing  is  finished.  Any 
of  the  following  methods  gives  good  results : 

a.  Stain    in    Mayer's    hsm-alum    six    hours   or  over   night, 


io6 


Methods  in  Plant  Histology 


wash  in  water  one  or  two  hours,  and  transfer  to  lo  per  cent, 
glycerine. 

b.  Stain  in  Delafield's  haematoxylin  thirty  minutes,  wash  in 
water  one  or  two  hours,  decolorize  in  water  acidulated  with 
hydrochloric  acid   (3    drops  of   HCl  to  100  cc.  water)   one  to 


Fig.  S3,    Pteris  cretica,     X  250. 

A ,  early  stage  in  the  development  of  the  archegonium.  5,  later  stage  showing  the  oosijhere,  ventral 
canal  cell,  and  three  nuclei  in  the  neck  canal.  C,  still  later  stage  almost  ready  for  fertilization.  The 
ventral  and  neck  canal  cells  are  breaking  down,  and  the  oosphere  is  nearly  mature.  Cells  surrounding  the 
oosphere  have  become  richer  in  protoplasmic  contents,  and  stain  more  deeply.  D,  first  division  of  the 
embryo.  E,  young  embryo  still  showing  the  outlines  of  the  four  quadrants.  The  apical  cell  in  the  lower 
left  cjuadrant  has  cut  off  the  first  layer  of  the  root  cap.  All  drawn  from  material  stained  in  bulk  in  alum 
carmine,  a  method  not  to  be  recommended. 

thirty  minutes  —  the  time  can  be  determined  only  by  experi- 
ment—  wash  in  water  until  the  rich  purple  color  of  the  haema- 
toxylin replaces  the  red  due  to  the  acid,  and  then  place  in  lo  per 
cent,  glycerine. 

c.  Stain  \Vith  Delafield's  haematoxylin  as  in  b,  and  after  the 
last  washing  in  water  stain  two  to  four  minutes  with  an  aqueous 


Pteridophytes  107 

solution  of  eosin,  wash  thoroughly  in  water,  and  transfer  to  the 
10  per  cent,  glycerine.  Instead  of  using  the  acid  alcohol  after 
the  haematoxylin,  the  eosin  may  be  allowed  to  act  for  several 
hours,  and  then  i  per  cent,  acetic  acid  may  be  used  for  a  few 
minutes.  Wash  very  thoroughly  in  water,  and,  if  the  stains 
appear  satisfactory,  transfer  to  10  per  cent,  glycerine  as  before. 

d.  Use  a  2  per  cent,  solution  of  iron  alum  two  hours,  wash 
in  water  five  minutes,  stain  in  )^  per  cent,  haematoxylin  two  to 
six  hours,  wash  in  water  five  minutes,  and  then  treat  again  with 
iron  alum  until  the  stain  is  sat- 
isfactory. Wash  thoroughly  in 
water  and  transfer  to  10  per 
cent,  glycerine,  which,  as  usual, 
will  concentrate  sufficiently  for 
mounting  in  three  or  four  days. 

For  paraffin   sections   such  A   '    ^""'^  B 

\  •        /•  J  Fig.  S4.     Pteris  cretica. 

as  are  shown  m  Ap-5.  5?  and  5</,  .      ,        ,  ,     ..      ,     . 

J  a       ~i^                ~r  I  ^,  section  of  a  nearly  mature  anthendium  snowing 

the    material    should    be    passed  the  antherozoids  inside      X  333.     .S,  a  developing 

r  antherozoid   showing   the  blepharoplast    drawn  out 

tVirnno-h    thf»    alrnhnlQ     allnwincr  '°*°  ^  deeply  staining  band.     X  1900.     Fixed  in 

tnrOUgn    tne    aiCOnOlS,   ailOWmg  chromo-acetic  add  and  stained  in  Haidenhain's  iron 

about   three  or  four  hours  for  -'"-h— 'oxyiin. 

each  grade.  The  mixtures  of  xylol  and  absolute  alcohol  should 
take  about  six  hours,  and  as  soon  as  the  pure  xylol  has  been 
added,  the  piece  of  paraffin  may  be  added  at  the  same  time.  In 
the  bath  two  or  three  hours  will  give  good  results.  It  would  be 
worth  while  to  determine  the  duration  of  the  bath  for  such  objects. 
Some  workers  claim  that  ten  or  fifteen  minutes  is  amply  suffi- 
cient, and  that  there  is  less  danger  from  shrinking,  while  others 
think  that  several  hours  is  better,  and  that  two  or  three  days  will 
do  no  damage,  if  the  fixing  has  been  thorough  and  the  tempera- 
ture is  not  allowed  to  become  higher  than  50°  C.  If  bergamot 
oil  be  used  instead  of  xylol,  there  is  less  danger  of  collapse  pre- 
vious to  the  bath,  but  the  bath  itself  must  usually  be  more  pro- 
longed, since  the  bergamot  oil  is  not  so  easily  gotten  rid  of  as  is 
the  xylol. 

For  morphological  purposes  sections  15  /i  to  20  /^  thick  are 
better  than  thinner  ones.    Delafield's  haematoxylin,  with  or  without 


io8 


Methods  in  Plant  Histology 


a  counter-stain  with  erythrosin,  is  good  for  such  sections  as 
are  represented  m  fig.  §j.  If  very  thin  sections  are  wanted  for 
cytological  study,  it  is  better  to  use  the  safranin-gentian  violet- 
orange  combination.  For  such  views  of  antheridia  as  are  shown 
in  fig.  5^,  Haidenhain's  iron  alum-haernatoxylin  seems  to  bring 
out  the  blepharoplast  (centrosome)  most  sharply. 

The  Sporophyte. — Methods  for  young  sporophytes  like  those 
shown  in  fig.  §j,  D  and  E,  are  the  same  as  for  archegonia  and 
antheridia. 

Roottips  to  show  the  prominent  apical  cell  are  easily  imbedded 
in  parafifin.     They  should  be  cut  i  5  ft  to  20  /a  thick.    Delafield's 

haematoxylin,  without  any  con- 
trast stain,  is  best  for  bringing 
out  the  prominent  apical  cell  and 
its  segments. 

Sporangia  should  also  be  cut 
m  parafifin.  Pteris  is  a  good  form 
for  sporangia,  since  the  long  mar- 
gmal  sorus  makes  it  possible  to 
get  an  immense  number  of  median 
longitudinal  sections  of  sporangia 
in  a  single  preparation.  {^Fig.  jj.) 
Pteris  ere  tic  a  can  always  be 
found  in  fruit  in  greenhouses. 
Select  a  series  of  stages.  The  leaf  should  be  cut  with  a  razor  — 
not  with  scissors  —  into  pieces  about  one-fourth  of  an  inch  long. 
If  sections  of  the  whole  leaf  are  not  wanted,  only  the  marginal 
sorus  need  be  cut  off;  in  this  way  a  much  greater  number  of 
sporangia  may  be  gotten  upon  a  slide.  Aspidium  and  Cyrtomium 
give  beautiful  views  of  the  indusium  covering  the  cluster  of 
sporangia.  The  most  satisfactory  preparations  will  be  gotten 
from  material  in  which  the  sporangia  have  not  yet  begun  to 
turn  brown. 

Botrychium  furnishes  excellent  and  usually  accessible  material 
for  studying  the  development  of  sporangia  of  the  eusporangiate 
type.     For  the  archesporium  and  early  stages  in  the  development 


Fig.  55. 
A ,  young  ; 


Pteris  cretica      X  560 


gc  in  the  development  of  the  spo- 
rangium. B,  older  stage  showing  the  tapetum. 
Fixed  in  chromo-acetic  acid  and  stained  in 
Delafield's  haematoxylin  and  erythrosin. 


Pteridophytes  1 09 

of  the  sporangia  the  material  should  be  collected  in  the  latter 
part  of  August  or  in  September.  When  the  sporangia  for  one 
year  are  ready  to  shed  their  spores,  the  sporangia  for  the  next 
year  will  be  found  in  early  stages  of  development.  When  a 
frond  is  found,  dig  the  plant  up  very  carefully  and  remove  the 
large  frond.  At  its  base  will  be  found  the  frond  for  the  next 
season,  the  sterile  portion  bent  over  so  that  its  tip  is  directed 
downward  and  the  fertile  portion  —  one-fourth  to  three-fourths 
of  an  inch  in  length  —  projecting  from  the  ventral  surface  of  the 
sterile  frond  and  directed  upward.  It  is  worth  while  to  preserve 
the  whole  bud  and  also  portions  of  the  stipe,  rhizome,  and  roots. 
These  vegetative  parts  should  be  cut,  with  a  razor  or  very  sharp 
knife,  into  pieces  about  one-fourth  of  an  inch  in  length.  The 
larger  roots  are  better  for  transverse  sections.  The  root  tips  are 
unusually  favorable  for  preparations  of  the  apical  cell.  The 
upper  part  of  the  rhizome  cuts  easily,  but  all  parts  of  this  plant, 
even  the  older  portions  of  the  rhizome,  can  be  cut  in  paraffin. 
Delafield's  haematoxylin  or  iron  alum-hsematoxylin  gives  fine 
preparations  of  the  young  sporangia.  The  older  sporangia,  from 
the  mother-cell  stage  up  to  the  shedding  of  the  spores,  stain 
better  in  cyanin  and  erythrosin  or  in  the  safranin-gentian  violet- 
orange  combination.  The  following  schedule  for  paraffin  sections 
will  give  elegant  mounts  of  the  root,  stipe,  and  rhizome : 

1 .  Stain  in  safranin,  about  24  hours. 

2.  Fifty  per  cent,  alcohol  until  little  or  no  safranin  is  left  in  the  cellulose 
walls.     Use  a  very  weak  acid  alcohol,  if  necessary. 

3.  Delafield's  haematoxylin,  5  to  10  minutes. 

4.  Water,  15  minutes. 

5.  Thirty-five  and  50  per  cent,  alcohol,  10  seconds  each. 

6.  Acid  alcohol  —  the  same  as  was  used  for  the  safranin  —  a  few  seconds. 

7.  Seventy  per  cent,  alcohol  until  the  purple  color  returns.  The  lignified 
walls  should  now  show  a  brilliant  red  and  the  cellulose  walls  a  rich  purple. 
If  either  stain  is  too  deep  or  too  faint,  do  not  proceed  any  farther  before 
making  the  necessary  correction.  If  the  safranin  washes  out,  and  the 
haematoxylin  is  too  intense,  do  not  use  any  acid  alcohol  in  the  second 
step  and  shorten  the  period  in  the  haematoxylin. 

8.  Eighty-five  and  95  per  cent,  alcohol,  a  few  seconds  each. 

9.  Hundred  per  cent,  alcohol,  30  seconds  to  i  minute. 

10.  Xylol  until  cleared. 

11.  Balsam. 


no 


Methods  m  Plant  Histology 


The  spore  mother-cells  of  Osmunda  are  excellent  for  a  study 
of  mitosis.  The  young  sporangia  of  0.  cinnamomea  and  0.  Clayto- 
niana  show  the  mother-cell  stage  in  the  autumn,  but  the  division 
into  spores  does  not  occur  until  the  following  spring,  in  the 
vicinity  of  Chicago  the  mitotic  figures  being  found  during  the 

latter  part  of  April.      0. 
P    9e^^Xi:iW^!/^^^^^i^'''i^i':'^^isJ^~  'j.  regalis  does  not  reach  the 

mother-cell  stage  in  the 
autumn.  Material  for 
mitosis  should  be  col- 
lected during  the  first 
two  weeks  in  May.  The 
material  may  be  fixed  in 
the  medium  chromo- 
acetic  solution  or  in  Flem- 
ming's  weaker  solution. 
Sections  should  not  be 
thicker  than  lO  /l*,  and 
5  /*  will  be  found  more 
satisfactory. 

Preparations  of  the 
woody  structure  of  the 
sporophyte  are  easily 
made.  The  rhizome  of 
Pteris  aquili?ia  affords  as 
good    material    as    any 

Fig.  56.     P.eris  aquilina.  {fig-S^^)'        I"  digging  Up 

A  part  of  a  transverse  section  of  the  vascular  bundle  of  the  rhizOmCS,    do   not    merely 
rhizome.     X  i66.     ^,  endodermis.    /,  pericycle.     j/.  sieve  tube,  _  ^ 

/,  scalariform  tracheid.  Drawn  from  a  celloidin  section  from  difif  doWU  Until  thc  rhi- 
material  fixed  in  picro-acetic  acid  and  stained  in  safranin  and  ° 

Deiafieid's  hsematoxyiin.  zomc  Can  be  graspcd  and 

then  pull  it  up,  for  such  material  is  sure  to  show  the  pericycle  of  the 
bundles  torn  away  from  the  parenchyma.  Dig  carefully  around 
the  rhizome  and  then  cut  off  with  a  very  sharp  knife  pieces  about 
two  inches  in  length.  Put  the  fresh  rhizome  into  the  hand  micro- 
tome and  cut  as  thin  sections  as  possible.  Keep  the  knife  wet 
with  95  per  cent,  alcohol  and  put  the  sections  into  95  per  cent. 


Pteridophytes 


alcohol  as  fast  as  they  are  cut.     Fifteen  or  twenty  minutes  is 

sufficient  for  fixing.     Pass  down  through  the  grades  of  alcohol, 

about  two  or  three  minutes  in  each   grade.      Stain   in   safranin 

twenty-four  hours,  wash  in  water  ten  minutes,  and  then  stain  in 

Delafield's  haematoxylin  ten  to  twenty  minutes.       Pass  through 

the  alcohols,  about  one   minute  in  each   grade,  at    70  per  cent, 

alcohol  transferring  to  acid  alcohol  from   one   to  five  seconds  ; 

then  pass  on,  clear  in 

xylol,  and    mount    in 

balsam.     If  the  stain 

is    successful,    the 

xylem  should  show  a 

brilliant  red,  and  the 

cellulose  walls  a  rich 

purple. 

Another  method  is 
to  stain  for  about  twen- 
ty minutes,  or  several 

,  ....  Fig.  57.     Marsilea  quadrifolia.     X  333. 

hours,  m  lodme  green      ^  ,  •  ^     i.      •         .  •  •     i.    •    u 

'                              o  A,  apex  of  megaspore  with  archegonium  containing  the oosphere. 

r>r  mf»fVi-\7l    crrp>f»n     rincf  Large  starch  grains  are  shown  beneath  the  archegonium.    ^,young 

ui    iiiCLH_yi  giccii,  iiiisc  gn^bryo.     Both  fixed  in  picro- acetic  acid  and  stained  in  Delafield's 

in  70   per  cent,  alcohol  ^hematoxylin.     (M.ss  M.  E.  Tarrant.) 

until  the  green  is  prominent  only  in  the  xylem,  then  stain  for 
from  one  to  three  minutes  in  acid  fuchsin  (i  per  cent,  solution 
in  70  per  cent,  alcohol),  dehydrate  rapidly,  clear  in  xylol,  and 
mount  in  balsam.  The  xylem  should  have  a  sharp  bright  green 
color,  and  the  cellulose  a  bright  red. 

The  most  beautiful  preparations  may  be  obtained  by  imbed- 
ding in  celloidin  and  staining  in  safranin  and  Delafield's  haema- 
toxylin. These  stains  allow  a  use  of  acid  which  extracts  all  color 
from  the  celloidin  and  still  leaves  a  sufficient  amount  in  the 
tissues.  The  large  apical  cells  of  rhizomes  are  easily  cut  in 
either  paraffin  or  celloidin. 

The  megaspores  and  microspores  of  Marsilea  are  easily 
obtained  {jigs.  57  and  58^.  Cut  away  a  portion  of  the  hard 
sporocarp  and  place  the  sporocarp  in  a  dish  of  water.  The 
gelatinous  ring  with  its  sori  will  sometimes  come  out  in  a  few 


Fig.  57.     Marsilea  quadrifolia. 


12 


Methods  i?i  Plant  Histology 


minutes.  In  less  than  twenty-four  hours  the  microspores,  start- 
ing from  the  one-cell  stage,  will  produce  the  mature  antherozoids. 
The  development  of  the  megaspore  is  equally  rapid.  Embryos 
are  abundant  in  two  or  three  days.  For  morphological  work, 
picro-acetic  acid,  used  hot,  is  very  good,  since  the  material  does 
not  occasion  so  much  difficulty  in  cutting.  Chromo-acetic 
material  allows  better  staining,  but  the  cutting  is  more  uncertain. 

It  is  best  to  prick  the 
megaspores  with  a 
needle  while  they  are 
in  the  fixing  fluid,  in 
order  to  facilitate  the 
infiltration  of  paraffin. 
Better  mounts  of  the 
microspores  can  be 
obtained  if  the  trou- 
blesome megaspores 
be  picked  out  from 
the  sorus  while  the 
material  is  still  in  the  fixing  agent  or  the  alcohols.  The  mega- 
spores must  be  imbedded  in  rather  hard  paraffin,  and  one  must 
expect  to  hone  the  knife  thoroughly  before  it  can  be  used  again, 
for  when  the  knife  strikes  a  megaspore  of  one  of  the  heterospor- 
ous  pteridophytes,  it  seems  like  striking  a  grain  of  sand. 

The  following  schedule  will  usually  give  good  sections  of  the 
hard  megaspores  of  the  heterosporous  pteridophytes,  whether 
they  are  to  be  cut  separately  or  in  their  sori  or  strobili.  The 
essential  features  of  the  method  are  suggested  by  Miss  F.  M. 
Lyon's  work  on  Selaginella: 

I.  Chromo-acetic  acid,  medium  solution,  2  to  6  days. 

2..  Wash-  in  water,  i  day. 

3.  Thirty-five  to  95  per  cent,  alcohol,  i  day  each. 

4.  One  hundred  per  cent,  alcohol,  3  or  4  days,  changing  several  times. 

5.  Mixtures  of  alcohol  and  xylol,  2  days. 
•6,  Pure  xylol  at  53°  C,  i  or  2  days. 

7.    Add  parafifin  to  the  xylol  and  keep  at  53°  C.  for  2  or  3  days. 


Fig.  58.     Marsilea  quadrifolia.     X  560. 

A ,  microspore  before  germination.  B,  microspore  with  antheridia 
nearly  mature.  Fixed  in  chromo-acetic  acid  and  stained  in  safranin- 
gentian  violet-orange. 


Pteridophytes  113 

8.  Pure  parafifin  53°  C,  3  or  4  days,  and  then  in  harder  paraffin  at  60°  C.  to 
70°  C,  2  or  3  days. 

9.  Imbed  in  rather  thin  cakes. 

While  the  method  is  tedious,  it  is  worth  the  trouble,  for  even 
old  strobili  of  Selagiiiella  yield  smooth  ribbons  at  5/x. 

^s  fig.  ^y  suggests,  the  mature  archegonia,  and  especially  the 
young  embryos,  may  be  removed  from  the  top  of  the  megaspore 
and  cut  with  perfect  ease. 

The  spermatozoid,  which  in  Marsilea  has  an  unusually  large 
number  of  turns  in  the  spiral,  may  be  mounted  by  methods 
already  described. 


CHAPTER  XVI. 

PTERIDOPHYTES. 
EQUISITINEJE. 

The  prothalHa  of  Equisetum  are  easily  grown  by  the  method 
already  described  for  the  Filicineae,  but  the  spores  must  be 
sown  as  soon  as  ripe,  because  they  fail  to  germinate  if  kept  more 
than  a  few  days.  In  the  vicinity  of  Chicago  the  spores  of 
Equisetum  arvense  are  shed  during  the  latter  part  of  April.  The 
methods  for  preparing  the  prothallia  are  the  same  as  for  the 
Filicineae.  The  sporangia  are  harder  to  cut,  but  good  prepara- 
tions should  be  secured  from  paraffin  material.  E.  arvense  is 
abundant  everywhere,  and  is  to  be  preferred  on  account  of  the . 
comparative  ease  with  which  the  sporangia  and  other  portions  of 
the  fertile  shoot  can  be  cut.  Longitudinal  sections  of  the 
younger  strobili  show  various  stages  in  the  development  of  the 
spores,  the  more  advanced  stages  being  found  at  the  base  of  the 
strobilus.  Tetrads  may  be  found  at  the  base  of  the  strobilus, 
while  the  spore  mother-cells  at  the  apex  are  still  undivided.  Of 
course,  it  is  impossible  to  stain  a  longitudinal  section  of  such  a 
strobilus  so  that  all  stages  will  be  satisfactory.  For  the  beginner, 
at  least,  this  is  not  a  serious  objection,  for  he  will  be  almost  sure 
to  secure  some  stage  beautifully  stained.  The  experienced 
worker,  who  is  able  to  control  his  staining  with  more  precision, 
will  prefer  transverse  sections.  Material  for  sporangia  should 
be  obtained  as  soon  as  the  fertile  shoot  appears  above  ground, 
and  if  it  can  be  obtained  earlier,  so  much  the  better.  When  the 
spores  are  shed,  the  young  sporangia  which  are  to  develop  the  next 
year  can  already  be  detected.  The  safranin-gentian  violet-orange 
combination  can  be  recommended  for  the  development  of  the 
mother-cell  and  the  formation  of  tetrads,  but  Delafield's  haema- 
toxylin  or  Haidenhain's  iron  alum-haematoxylin  will  be  more 
satisfactory  for  earlier  stages. 

"5 


Ii6  Methods  in  Plant  Histology 

The  roots  are  very  small,  but  have  large  cells  and  easily  yield 
good  preparations.  In  case  of  such  small  objects  it  is  a  good 
plan  to  add  a  few  drops  of  eosin  to  the  alcohol  during  the  pro- 
cess of  dehydrating,  in  order  that  the  material  may  be  seen  more 
easily.  The  slight  staining  does  no  damage,  even  if  more 
critical  stains  are  to  be  used  after  the  sections  are  cut.  It  is 
easy  to  get  longitudinal  sections  of  the  roots  by  cutting  trans- 
verse sections  of  the  nodes.  In  E.  arvense  these  roots  at  the  nodes 
are  quite  numerous.  Sections  of  the  stem  of  the  fertile  shoot  of 
E.  arvense  are  easily  cut  in  paraffin  or  celloidin,  but  sections  of 
the  stem  of  E.  hiemale  or  similar  species  do  not  cut  in  paraffin, 
and  results  are  rather  uncertain  even  in  celloidin.  The  growing 
points  of  stems,  however,  may  be  cut  with  ease  in  paraffin.  E. 
arveme  is  particularly  favorable  on  account  of  the  numerous 
apical  cells  which  may  be  found  in  a  single  preparation.  Dela- 
field's  haematoxylin,  used  alone,  is  good  for  the  apical  cells,  but 
for  sections  of  older  stems  a  slight  counter-stain  with  erj'throsin 
will  improve  the  mount. 


CHAPTER  XVII. 


PTERIDOPHYTES. 
LYCOPODINEiE. 

It  is  not  very  difficult  to  get  paraffin  sections  of  young 
sporangia  of  Selaginella,  but  the  method  just  recommended  for 
Marsilea  should  be  resorted  to  for  the  older  strobili  {fig.  5p). 

The  older  megaspores  had  better  be  pricked  with  a  needle 
and  cut  one  at  a  time.  A  slight  puncture  at  the  basal  portion  of 
the  megaspore 
does  no  damage 
and  insures  a  thor- 
ough infiltration. 
If  the  megaspores 
are  imbedded  sep- 
arately, they  will 
usually  orient 
themselves  so  that 
sections  perpen- 
dicular to  the  par- 
affin cake  will 
show  the  most  in- 
structive views  of 
the  gametophyte 
structures. 


Fig.  59.     Selaginella  Mertensii.     X  93. 


Ay  microsporangium  containing  microspoi 
ing  the  beginning  of  the  prothallium.     FixeU  ni   p 
Delafield's  hae-    stained  in  Delafield's  hsematoxylin.     (Miss  M.  E.  Tarrant.) 


a  megaspore  show- 
icro-acetic  acid   and 


matoxylin,  used  alone,  is  a  good  stain.  The  safranin-gentian 
violet-orange  combination  gives  a  brilliant  differentiation  in 
stages  likey?^.  jp,  B.  The  microspores  of  Isoetes  offer  the  same 
difficulties.  Cyanin  and  erythrosin  is  a  fine  combination  for  the 
reserve  food-stuffs  in  Isoetes  macrospores. 

For  young  sporangia  of  Isoetes,  the  leaves  should  be  cut  off 
about  one-eighth  of  an  inch  above  the  sporangia,  and  the  stem 

117 


Ii8  Methods  in  Plaiit  Histology 

should  be  cut  off  below,  leaving  just  enough  to  hold  the 
sporangia  together.  Cut  longitudinal  sections  and  stain  in 
Delafield's  haematoxylin,  with  or  without  the  addition  of  a  light 
touch  of  erythrosin.  The  older  sporangia  had  better  be  removed 
and  cut  separately.  Transverse  sections  of  the  stem  are  very 
interesting.  Young  stems  and  the  apices  of  old  ones  cut  well 
in  paraflfin,  but  older  stems  will  give  more  satisfactory  results  in 
celloidin. 

It  is  much  easier  to  get  good  preparations  of  the  sporangia 
of  Lycopodium,  since  there  are  no  megaspores  with  their  hard 
walls.  Delafield's  haematoxylin,  without  any  contrast  stain,  will 
bring  out  the  developing  sporangia,  which  are  usually  to  be 
found  even  after  the  sporangia  in  the  lower  part  of  the  strobilus 
are  beginning  to  shed  their  spores.  The  directions  for  the 
rhizome  of  the  Filicineae  will  also  serve  for  the  stem  of 
Lycopodium. 


CHAPTER  XVIII. 

SPERMATOPHYTES. 

In  this  immense  group  we  cannot  hope  to  give  even  approxi- 
mately complete  directions  for  making  preparations,  but  must 
be  content  to  give  a  few  hints  which  may  prove  helpful  in  col- 
lecting material  and  in  securing  mounts  of  the  more  important 
structures  of  the  flowering  plants.  We  shall  consider  the  gym- 
nosperms  and  the  angiosperms  separately,  although .  in  many 
respects  the  technique  is  the  same  for  both. 

GYMNOSPERMS. 

Since  Pinus  is  a  characteristic  type,  we  shall  describe  methods 
for  demonstrating  various  phases  in  the  life-history  of  this  genus, 
hoping  that  the  directions  will  enable  the  student  to  experi- 
ment intelligently  with  other  forms. 

Spermatogenesis. —  In  October  the  clusters  of  staminate  cones 
which  are  to  shed  their  pollen  in  the  coming  spring  are  already 
quite  conspicuous.  The  cones  should  be  picked  off  separately, 
and  the  scales  should  be  carefully  removed  so  as  to  expose  the 
delicate  greenish  cone  within.  At  this  time  the  archesporial 
cells  are  easily  distinguished.  Material  collected  in  January,  or 
at  any  time  before  growth  is  resumed  in  the  spring,  shows  about 
the  same  stage  of  development.  If  it  is  desired  to  secure  a 
series  of  stages  with  the  least  possible  delay,  a  branch  bearing 
numerous  clusters  of  cones  may  be  brought  into  the  laboratory 
and  placed  in  a  jar  of  water.  Growth  is  more  satisfactory  in 
case  of  branches  broken  off  in  the  winter  than  in  those  brought 
in  before  there  has  been  any  period  of  rest.  The  material  can 
be  examined  from  time  to  time,  and  a  complete  series  is  easily 
secured.  The  karyokinetic  figures  in  the  pollen  mother-cells 
furnish  exceptionally  instructive  preparations.  Staminate  cones 
which  will  yield  karyokinetic  figures  can  be  selected  with  con- 
siderable certainty  by  examining  the  fresh  material.     Crush  a 

119 


120  Methods  in  Plant  Histology 

microsporangium  from  the  top  of  the  cone  and  one  from  the 
bottom,  add  a  small  drop  of  water  and  a  cover  to  each,  and  exam- 
ine. If  there  are  pollen  tetrads  at  the  bottom,  but  only  undi- 
vided spore  mother-cells  at  the  top,  it  is  very  probable  that 
longitudinal  sections  of  the  cone  will  yield  the  figures.  If  a  drop 
of  methyl  green  be  allowed  to  run  under  the  cover,  it  will  enable 
one  to  see  whether  figures  are  present  or  not.  When  desirable 
cones  are  found,  they  should  be  cut  longitudinally  into  two 
halves.  The  later  stages,  showing  the  germination  of  the  micro- 
spores, furnish  better  sections  if  the  cones  are  cut  transversely 
into  small  pieces  about  three-sixteenths  of  an  inch  thick.  It  is 
very  easy  to  get  excellent  mounts  of  the  pollen  just  at  the  time  of 
shedding.  Shake  a  large  number  of  cones  over  a  piece  of  paper, 
thus  securing  an  abundance  of  material.  Fix  in  chromo-acetic 
acid,  wash  in  water  (a  few  minutes  is  sufficient,  and  the  water  need 
not  be  changed),  pass  through  the  alcohols,  allowing  each  to 
act  for  about  two  hours,  make  the  usual  gradual  transition  from 
alcohol  to  xylol,  and  from  xylol  to  paraffin.  It  is  best  that  the 
material  should  be  in  a  small  bottle  not  more  than  one-fourth 
of  an  inch  in  diameter ;  at  any  rate,  the  pollen  should  be  in  such 
a  bottle  during  infiltration,  which  should  not  require  more  than 
two  or  three  hours,  although  a  longer  period  does  no  harm  if  the 
temperature  does  not  rise  above  52°  or  53°  C.  Now  put  the  lower 
portion  of  the  bottle  into  cold  water,  and  thus  harden  the  paraffin 
as  quickly  as  possible.  Break  the  bottle  carefully,  cut  off  the  lower 
portion  of  the  paraffin  containing  the  pollen,  mount  it  on  a  block 
in  the  usual  manner,  and  trim  away  some  of  the  paraffin  so  that 
two  parallel  surfaces  will  make  the  sections  ribbon  well.  Material 
in  this  stage  shows  a  large  tube  nucleus,  a  somewhat  lenticular 
cell  with  a  more  deeply  staining  nucleus,  and,  lastly,  two  small 
prothallial  cells  quite  close  to  the  spore  wall.  The  prothallial 
cells  cannot  always  be  detected  at  this  stage,  and  there  may  be 
some  doubt  as  to  whether  two  such  cells  are  always  present. 
The  division  of  the  lenticular  cell  into  "stalk  cell"  and  "body 
cell,"  and  also  the  division  of  the  body  cell  into  the  two  male 
cells,  must  be  looked  for  in  sections  of  the  nucellus  of  the  ovule. 


Spermatophytes 


121 


Oogenesis. — The  entire  ovulate  cone  at  the  time  of  pollina- 
tion is  easily  cut  in  paraffin.  Longitudinal  sections  of  the  cone 
at  this  time  give  good  views  of  the  bract  and  ovuliferous  scale 
bearing  the  ovules.  The  integument  is  very  well  marked,  and 
in  the  nucellus  one  or  more  sporogenous  cells  can  usually  be 
distinguished.  As  soon  as  the  scales  close  up  after  pollination, 
the  cone  will  be  too  hard  to  cut,  and  it  will  be  necessary  to 
remove  the  scales  and  cut  them  separately.  For  a  study  of  the 
ovule  and  the   structures  within  it,  better  preparations  will   be 


^.<^- 


Jovcc  f\pp.  Nt*t. 

Fig.  6o.     Pinus  Laricio.     X  104. 

A,  top  of  prothallium  with  an  archegonium  just  before  the  cutting  off  of  the  ventral  canal  cell. 
Fixed  in  Flemming's  weaker  solution  and  stained  with  Haidenhain's  iron  alum-haematoxylin.  Collected 
June  18,  1897.  B,  C,  and  D,  early  stages  in  the  formation  of  the  embryo.  Fixed  in  chromo-acetic  acid, 
and  stained  in  safranin -gentian  violet-orange.     Collected  July  2,  1897. 

obtained  by  carefully  cutting  off  the  pair  of  ovules  from  the 
scale.  For  preparations  like  that  represented  in  fig.  60,  A,  it  is 
a  good  plan  to  remove  the  endosperm  with  its  archegonia  from 
the  ovule.  Fixing,  infiltration,  and  cutting  will  then  occasion 
but  little  trouble,  and  the  whole  ribbon  may  be  gotten  upon  a 
single  slide.  However,  at  this  stage  the  pollen  tubes  with  their 
contents  are  rapidly  working  their  way  through  the  nucellus 
toward  the  archegonia,  and  consequently  it  is  better  to  retain 


122  Methods  i?i  Plant  Histology 

enough  of  the  tissues  of  the  ovule  to  keep  the  nucellus  in  place. 
In  later  stages,  after  fertilization  has  taken  place,  it  is  necessary 
to  remove  the  endosperm.  In  stages  like  fig.  60,  B,  C,  D,  and 
later,  the  developing  testa  should  be  dissected  away  with  great 
care,  for  a  very  slight  pressure  is  sufficient  to  injure  the  delicate 
parts  within.  Mature  embryos  may  be  dissected  out  from  the 
endosperm  before  fixing,  but  it  is  hardly  necessary,  since  they 
cut  quite  well  if  left  in  place.  The  "pine  nuts"  or  "pinon,"  to 
be  found  upon  the  market,  are  good  for  a  study  of  the  mature 
embryo.  The  testa,  which  is  quite  a  hard  shell,  should  be  taken 
off,  and  the  endosperm  should  be  allowed  to  soak  in  water  for 
about  twenty-four  hours,  after  which  the  embryo  may  be  dis- 
sected out  and  fixed. 

The  period  at  which  the  various  stages  may  be  found  varies 
with  the  species,  the  locality,  and  the  season.  In  Pinus  Laricio 
(the  common  Austrian  pine)  at  Chicago,  in  the  season  of  1897, 
material  collected  May  27  did  not  yet  show  archegonia ;  the 
ventral  canal  cell  was  cut  off  about  June  21  (see  fig.  61),  the 
fusion  of  the  pronuclei  occurred  about  a  week  later,  and  stages 
like  fig.  60,  B,  C,  and  D,  were  common  in  material  collected  July 
2.  In  the  season  of  1896  all  the  stages  appeared  about  two 
weeks  earlier.  In  Pinus  sylvestris  the  stages  appeared  a  little 
earlier  than  in  Pinus  Laricio.  After  the  stage  shown  in  fig.  60,  A, 
has  appeared,  it  is  necessary  to  collect  at  intervals  of  not  more 
than  two  days  until  the  stage  shown  mfig.  60,  D,  is  reached.  If 
collections  are  made  at  intervals  of  four  or  five  days,  the  most 
interesting  stages,  like  the  cutting  off  of  the  ventral  canal  cell, 
fertilization,  and  the  first  divisions  of  the  nucleus  of  the  oospore, 
may  be  missed  altogether.  It  should  be  mentioned  that  all  the 
ovules  of  a  cone  will  be  in  very  nearly  the  same  stage  of  devel- 
opment. 

A  rather  strong  chromo-acetic  acid  (i  g.  chromic  acid  and 
1/2  cc.  glacial  acetic  acid  to  100  cc.  water)  can  be  recom- 
mended for  the  entire  scries  in  spermatogenesis,  oogenesis,  fer- 
tilization, and  formation  of  the  embryo.  After  repeated  trials 
the   popular  Flemming's   solution   does   not   seem  to  be  at  all 


Spennatophytes 


123 


superior  and  often  fails  to  give  as  good  results  as  the  cheaper 
fixing  agent. 

The  following  stains  may  be  suggested :  for  studying  the 
pollen  tubes  in  the  nucellus,  cyanin  and  erythrosin ;  for  the 
development  of  the  archegonium  up  to  the  stage  shown  in 
fig.  60,  A, 
Delafield's 
haematoxy- 
lin;  for  the 
stages  shown 
in  fig.  60,  A, 
iron  alum- 
haematoxylin 
or  the  safra- 
nin  -  gentian 
violet-orange 
combination; 
for  the  stage 
shown  in  fig. 
61,  nothing 
seems  to 
equal  the 
safranin-gen- 
tian  violet- 
orange  com- 
bination ;  for 
stages  like 
fig.  60,  B,  C, 
and  D,  and 
also  for  later 
stages  in  the 
development 
of  the  em- 
bryo, Delafield's  haematoxylin  brings  out  the  walls  perfectly,  but 
since  mitotic  figures  are  very  frequent  in  these  stages,  it  is  worth 
while  to  use  the  safranin  combination  with  some  preparations, 


1 

•    #«        ^- 

^  ^-  •  5  « 

#  "^ 

# 

1 

^    m  ^ 

'••S 

f 

^  ■  .♦  *^'  f ; 

A 

^ -ir    ■  v>,.i»_l^alMi 

d 

Fig.  61.     Pinus  Laricio.     X  710. 


The  mitotic  figure  concerned  in  cutting  off  the  ventral  canal  cell.  The 
nucleus  at  the  lower  end  of  the  spindle  is  the  nucleus  of  the  oosphore. 
Fixed  in  chromo-acetic  acid,  and  stained  in  the  safranin-gentian  violet- 
orange  combination.     Collected  June  21, 1897. 


124  Methods  in  Plattt  Histology 

although  it  is  much  inferior  to  Delafield's  hsematoxylin  when 
cellulose  walls  are  to  be  emphasized. 

All  the  stages  which  have  been  described  can  be  cut  in 
paraffin  with  little  difficulty. 

The  Leaves. — The  leaves  of  our  common  gymnosperms  cut 
readily  in  paraffin  while  they  are  young  and  tender,  but  as  they 
approach  maturity  it  is  a  fruitless  task  to  attempt  paraffin  sec- 
tions. Celloidin  sections  are  far  more  satisfactory.  Cut  the 
needles  into  pieces  about  one-fourth  of  an  inch  long,  fix  in  a 
picro- corrosive -acetic  mixture  {%  g.  picric  acid,  2  g.  cor- 
rosive sublimate,  i  cc.  glacial  acetic  acid,  lOO  cc.  50  per 
cent,  alcohol).  If  used  hot,  five  minutes  is  sufficient,  but  if 
used  cold  it  should  be  allowed  to  act  for  two  or  three  hours. 
After  the  material  has  been  imbedded  in  celloidin,  the  block 
should  be  placed  in  equal  parts  of  95  per  cent,  alcohol  and 
glycerine  for  a  few  days,  after  which  it  should  cut  quite  readily. 
Stain  with  safranin  and  Delafield's  haematoxylin,  clear  in  Eycle- 
shymer's  clearing  mixture,  and  mount  in  balsam. 

Fairly  good  sections  may  be  obtained  in  great  quantities  with 
little  trouble  by  the  following  method :  Make  a  bunch  of  the 
needles  as  large  as  one's  little  finger,  wrap  them  firmly  together 
with  a  string,  allowing  about  an  eighth  of  an  inch  of  the  bunch 
to  project  above  the  wrapping;  then  fasten  the  whole  in  a  hand 
microtome,  and  every  stroke  of  the  razor  will  give  twenty  or 
thirty  sections,  some  of  which  will  surely  be  good.  As  the  sec- 
tions are  cut,  they  maybe  put  directly  into  95  per  cent,  alcohol, 
and  after  a  few  minutes  can  be  transferred  to  50  per  cent,  alco- 
hol and  then  to  the  stain.  Dehydrate,  clear  in  xylol,  and  mount 
in  balsam. 

Stems  and  Roots. — With  a  sharp  razor  fairly  good  sections 
of  stems  and  roots  may  be  made  without  imbedding,  especially 
if  the  hand  microtome  be  used.  Young  buds  may  be  cut  in 
paraffin.  Stems  and  roots  as  large  as  half  an  inch  in  diameter 
can  be  cut  in  celloidin.  The  material  should  be  cut  into  pieces 
not  more  than  one-fourth  of  an  inch  long.  The  following  treat- 
ment should  give  good  results  : 


Spermatophytes  125 

1.  Picro-corrosive-acetic  mixture,  five  minutes  if  used  hot,  or 
wo  to  three  hours  if  used  cold. 

2.  Wash  in  50  per  cent,  alcohol,  to  which  a  little  iodine  has 
been  added,  two  hours;   70  per  cent.,  85  per  cent.,  95  per  cent., 

our  hours  each ;  absolute  alcohol,  ten  hours ;  then  change  to 
fresh  absolute  alcohol,  which  should  act  for  ten  hours  longer. 

3.  Ether  alcohol,  twenty-four  hours.  Some  prefer  to  pre- 
cede the  ether  alcohol  by  a  mixture  of  equal  parts  of  ether 
alcohol  and  absolute  alcohol. 

4.  Thin  celloidin  (about  2  per  cent.),  two  or  three  days; 
6  per  cent,  celloidin,  two  or  three  days;  10  per  cent,  celloidin, 
two  or  three  days.  After  the  thin  celloidin  has  acted  for  a  few 
days,  the  cork  may  be  removed  for  a  short  time  each  day,  thus 
allowing  the  thin  celloidin  to  become  thick  by  the  evaporation 
of  the  ether  alcohol. 

5.  Get  some  small  blocks  of  wood  (three-eighths  inch  cubes 
of  white  pine  are  good),  wet  one  of  them  in  ether  alcohol,  dip  it 
into  thin  celloidin,  place  the  object  upon  the  block  in  convenient 
position  for  cutting,  pour  over  it  a  few  drops  of  10  per  cent,  cel- 
loidin, and  then  plunge  the  whole  into  chloroform.  Leave  it  in 
the  chloroform  about  twenty-four  hours,  and  then  transfer  to  a 
mixture  of  isqual  parts  of  95  per  cent,  alcohol  and  glycerine, 
where  it  should  remain  for  several  days.  Material  may  be  kept 
here  indefinitely.  Even  refractory  stems  may  be  cut  after  they 
have  been  in  this  mixture  for  a  couple  of  weeks. 

6.  Cut  the  sections,  keeping  the  knife  wet  with  the  alcohol 
and  glycerine  mixture.  Transfer  the  sections  to  70  per  cent, 
alcohol,  then  to  50  per  cent.,  and  then  to  the  stain. 

7.  Stain  in  safranin,  twenty-four  hours;  wash  in  35  per  cent, 
alcohol,  about  a  minute  ;  stain  in  Delafield's  haematoxylin,  five  to 
ten  minutes;  wash  in  water,  two  minutes ;  35  per  cent,  alcohol,  two 
to  five  minutes ;  50  per  cent,  alcohol,  two  to  five  minutes ;  acid 
alcohol,  one  to  ten  seconds;  70  per  cent.,  85  per  cent.,  95  per 
cent.,  about  two  minutes  each ;  Eycleshymer's  clearing  mixture 
until  cleared,  usually  about  one  or  two  minutes.  Mount  in 
balsam. 


:26 


Methgds  m  Plant  Histology 


Xylem  should  show  a  brilliant  red  color  and  cellulose  a 
rich  purple,  if  the  stain  is  successful.  If  either  stain  is  too  weak 
or  too  prominent,  the  duration  of  the  stain,  the  length  of  time 
in  the  alcohols,  or  the  time  in  acid  alcohol  must  be  varied  until 
the  desired  result  is  secured. 

People  who  make  all  their  anatomical  sections  without  imbed- 
ding may  regard  this  method  as  tedious  and  unnecessary,  but 

such   preparations 


TWUwdtr^itM  °{  Chicago. 


i<x«9.mil,li>n3.ti,y. 
<a.  U/  THau^u.  nio. 


'M!mmmMmM>>Mmmm9* 


/oixr.  ;^pp.  A\i5.. 

' 

\ 

lea  ^aii-iM^ 

i     1 

i 

g 

s 

^^ 

',U/-A^A\.: 

will  show  much 
which  is  never  seen 
in  mere  free-hand 
sections,  for  the 
reason  that  free- 
hand sections,  if 
thin  enough  to 
show  any  detail, 
will  lose  most  of 
their  cell  contents, 
while  in  celloidin 
sections  every- 
thing is  held  in 
place.  Even  if  the 
celloidin  sections 
be  passed  through  absolute  alcohol  and  cleared  with  clove  oil,  a 
process  which  dissolves  away  the  celloidin,  the  contents  of  the 
cells  will  still  be  retained  in  most  cases,  and  stains  which 
cannot  be  extracted  from  celloidin  may  be  used.  However, 
we  prefer  to  use  the  safranin  and  Delafield's  haematoxylin  and 
retain  the  celloidin,  since  this  combination  can  be  extracted 
completely  from  the  celloidin  and  still  leave  the  object  brilliantly 
stained. 

For  preparations  of  the  mature  wood  a  piece  of  white  pine 
from  a  dry-goods  box  furnishes  perfect  material.  It  should  not 
be  imbedded,  but  the  cutting  w^ll  be  facilitated  if  the  piece  be 
soaked  for  a  few  hours  in  water  or  in  the  alcohol  and  glycerine 
mixture.     In  all  preparations  designed  to  show  the  structure  of 


Fig,  62. 
Slides  showing  labels  and  methods  of  arranging  sections. 


Spermatophytes  127 

wood  there  should  be  three  sections  —  a  transverse,  a  longitu- 
dinal radial,  and  a  longitudinal  tangential.  These  may  be 
arranged  as  in  the  upper  slide  oi  fig.  62,  but  if  it  is  desired  to 
make  a  thorough  study  of  the  structure,  it  is  a  good  plan  to  have 
on  each  slide  several  sections  of  each  kind,  thus  having  an 
opportunity  to  use  a  variety  of  stains.  Fuchsin  and  iodine  green 
is  a  good  combination.  The  safranin  and  Delafield's  hasma- 
toxylin  is  also  excellent. 

For  a  study  of  young  stems  or  roots,  preparations  like  that 
shown  in  the  lower  slide  oi  fig.  62  will  be  found  very  convenient. 


CHAPTER  XIX. 

SPERMATOPHYTES. 
ANGIOSPERMS. 

Success  depends  largely  upon  judgment  and  care  in  selecting 
and  trimming  material  before  it  is  put  into  the  fixing  agent  in 
the  field.  While  the  following  directions  cannot  be  applied  to 
all  plants,  they  should,  nevertheless,  enable  the  student  to  make 
such  modifications  as  may  be  demanded  by  any  particular  form 

Floral  Development. —  For  a  study  of  floral  development  very 
young  buds  are  necessary,  and  it  is  best  to  select  those  forms 
which  have  rather  dense  clusters  of  flowers,  in  order  that  a  com- 
plete series  may  be  obtained  with  as  little  trouble  as  possible. 

The  usual  order  of  appearance  of  floral  parts  is  (i)  calyx, 
(2)  corolla,  (3)  stamens,  and  (4}  carpels,  but  if  any  of  these 
organs  are  reduced  or  metamorphosed,  their  order  of  appearance 
may  be  affected. 

Floral  development  is  easily  studied  in  the  common  Capsella 
bursa-pastoris.  The  best  time  to  collect  material  is  late  in  March 
or  early  in  April.  Dig  up  the  plant,  carefully  remove  the  leaves, 
and  in  the  center  of  the  rosette  a  tiny  white  axis  will  be  found. 
A  series  of  these  axes  from  one-eighth  of  an  inch  to  three- 
eighths  of  an  inch  in  length  and  from  one-sixteenth  of  an  inch 
to  three-sixteenths  of  an  inch  in  diameter  will  give  a  very  com- 
plete series  of  stages  in  the  development  of  the  floral  organs. 
Preparations  from  the  apex  of  the  shoot  taken  after  the  inflores- 
cence appears  above  ground  are  not  to  be  compared  with  these 
taken  early  in  the  season.  Fix  in  chromo-acetic  acid  and  stain 
in  Delafields  haematoxylin.  The  sections  should  be  longitudinal 
and  about  5  /x  thick. 

The  common  dandelion,  Taraxacum  officinale,  affords  an  excel- 
lent series  with  little  labor.  Examine  vigorous  plants  which  have, 
as  yet,  no  flowers  or  buds  in  sight.  Dig  up  the  plant  and  dissect 
away  the  leaves.  If  there  is  a  white  cluster  of  flower  buds,  the 
'  129 


130  Methods  in  Plant.  Histology 

largest  not  more  than  three-sixteenths  of  an  inch  in  diameter, 
cut  out  the  cluster,  leaving  only  enough  tissue  at  the  base  to 
hold  the  buds  in  place.  Larger  heads  should  be  cut  separately. 
Fix  and  stain  as  in  Capsella. 

Our  most  common  thistle,  Cnicus  lanceolatus,  shows  the  floral 
development  with  unusual  clearness,  but  the  preparation  of  the 
material  is  somewhat  tedious.  The  involucre,  which  is  too  hard 
to  cut,  must  be  carefully  dissected  away.  Retain  only  enough  of 
the  receptacle  to  hold  the  developing  florets  in  place.  A  series 
of  sizes  with  discs  varying  from  one-eighth  of  an  inch  to  three- 
eighths  of  an  inch  in  diameter  will  show  the  development  from 
the  undifferentiated  papilla  up  to  the  appearance  of  the  arche- 
sporial  cell  in  the  nucellus  of  the  ovule.  The  Canada  thistle, 
C?iicus  arvensis,  is  equally  good,  but  it  is  more  diflficult  to  dissect 
out  the  desirable  parts. 

In  the  willows,  Salix,  the  bud  scales  must  be  removed  and 
the  copious  hairs  should  be  trimmed  off  as  much  as  possible 
with  scissors,  after  which  the  catkin  should  be  cut  in  two  longi- 
tudinally and  placed  in  the  fixing  agent. 

Spermatogenesis. — The  earlier  stages  in  spermatogenesis  will 
be  found  in  the  preparations  of  floral  development.  For  tracing 
the  nuclear  changes  involved  in  this  process  the  lilies  furnish 
very  good  material,  because  the  cells  and  nuclei  are  exceptionally 
large.  Several  species  of  Lilium  are  common  in  greenhouses, 
and  these  may  be  used  where  wild  material  is  not  available.  In 
early  stages  where  the  sporogenous  cells  have  not  yet  begun  to 
round  off  into  spore  mother-cells  it  is  sufificient  to  remove  the 
perianth,  retaining  just  enough  of  the  receptacle  to  hold  the  sta- 
mens in  place.  Transverse  sections  show  the  six  stamens  and 
also  the  young  ovary.  After  the  spore  mother-cells  have  begun 
»to  round  off,  each  stamen  should  be  removed  so  as  to  be  cut 
separately.  It  is  very  desirable  to  secure  stamens  showing  the 
mitotic  figures  which  occur  during  the  division  of  the  spore 
mother-cell  into  the  four  microspores.  Since  the  pollen  mother- 
cells  are  apt  to  be  in  approximately  the  same  stage  of  develop- 
ment  throughout  the    anther,   it  is  worth   while    to   determine 


Spermatophytes 


131 


whether  mitotic  figures  are  present  before  putting  the  material 
into  the  fixing  agent.  The  procedure  indicated  for  Pi?ius  can  be 
followed  here.  It  is  not  necessary  to  cut  the  stamens  into 
pieces  before  fixing,  since  they  are  easily  penetrated  and  infil- 
trated ;  in  later 
stages  the  sta- 
mens must  not  be 
cut  into  pieces, 
since  the  pollen 
grains  are  easily 
washed  out. 

Transverse 
sections  are  bet- 
ter for  morpho- 
logical purposes; 
but  where  noth- 
ing is  desired  ex- 
cept thedevelop- 
ment  of  pollen 
grains  from  the 
spore  mother- 
cells,  much  more 
material  can  be 
gotten  under  a 
cover  by  using 
longitudinal  sec- 
tions. 


Fo  r  e  a  r 1 


A^    Salix   tristis.     X  694.     Transverse   section   of   a  portion  of  a  young 


StaffeS   UD   to   that     ^"'l^^''   showing   large   sporogenous  cells,  one  layer  of  tapetal  cells  (more 
o  r  deeply  shaded  in  the  figure) .  and  ' 


from  three  to  four  layers  of  wall  cells. 
Salix  petiolaris.  X  594.  Small  portion  of  transverse  section  of  a  nearly 
mature  anther  showing  five  pollen  grains,  two  tapetal  cells,  and  two  layers 
of  wall  cells.  C,  Lilium  auratum.  X,505.  Section  of  a  pollen  grain  show- 
ing the  large  tube  nucleus  and  two  smaller  generative  nuclei.  Fixed  in 
chromo-acetic  acid  and  stained  in  safranin-gentian  violet-orange.  D, 
Lilium  tigrinum.  X  505,  Pollen  grain  showing  tube  nucleus  in  the  mid- 
dle and  a  lenticular  cell  with  the  generative  nucleus  at  one  end  of  the  grain. 


shown  in  fig.  6j 

A;  D,elafield's 

haematoxylin     is 

very  satisfactory; 

in  stages  like  B,  C,  and  D  of  the  same  figure,  cyanin  and  eryth- 

rosin  often  give  excellent  differentiation,  the  tube  nucleus  taking 

the  erythrosin,  and  the  generative  nucleus  the  cyanin,  while  the 


132 


Methods  in  Pla?it  Histology 


starch  grains  which  are  often  abundant  at  this  stage  take  a  faint 
pink  from  the  erythrosin.  However,  the  safranin-gentian  violet- 
orange  combination  shows  nuclear  details  to  better  advantage, 
and  readily  distinguishes  the  tube  and  generative  nuclei,  although 
it  does  not  give  such  a  striking  color  contrast  as  the  cyanin  and 

erythrosin.  The  starch  grains 
are  brilliantly  stained  by  the 
gentian  violet. 

The  mature  pollen  grain, 
after  shedding,  may  be  pre- 
pared by  the  method  already 
described  for  Pijius. 

Oogenesis.  —  As  in  sper- 
matogenesis,  the  early  stages 
will  be  found  in  preparations 
of  floral  development.  The 
origin  and  development  of  the 
macrospore  are  easily  traced 
in  Lilium.  In  very  young 
stages,  before  the  appearance 
of  the  integument,  the  ovary 
may  be  removed  from  the 
flower  and   placed  directly  in 

;ad  of  Aster.    5, pod  of  Capsella.    C,  transverse    the   fixing    agent,  but    in    later 
of  ovary  of  Lilium.    The  dotted  lines  show  how  "        ° 

the  material  should  be  trimmed  before  fixing,  StagCS,   SUch    aS    are    shown    in 

figs.  66-^1,  strips  should  be  cut  off  from  the  sides  of  the  ovary 
in  order  to  secure  more  rapid  fixing  and  more  perfect  infiltration 
with  paraffin.  The  dotted  lines  in  fig.  64,  C,  show  about  how  much 
should  be  cut  off.  This  is  a  much  better  plan  than  to  secure 
rapid  fixing  and  infiltration  by  cutting  the  ovary  into  short 
pieces,  because  the  ovules  will  be  in  about  the  same  st^ge  of 
development  throughout  the  ovary,  and  when  one  finds  desirable 
stages  like  those  from  which  these  photomicrographs  were  taken, 
it  is  gratifying  to  have  these  pieces  as  long  as  possible. 

In  lilies,  and  other  forms  with   large  cells,  the   entire  series 
shown   in  figs.  65-yo  may  be  fixed   in  chromo-acetic  acid  and 


A  ,  head 
section  of  ovary 


Spennatophyte 


33 


stained  in  safranin-gentian  violet-orange,  but  stages  earlier  than 
that  shown  m  fig.  65  are  more  satisfactory  if  stained  in  Delafield's 
haematoxylin.  Stages  like  that  shown  in  fig.  66  and  also  those 
between  ^^.  66  and  fig.  6'j  are  the  most  difficult  to  stain,  and 
only  the  utmost  care  and  patience  will  insure  first-class  prepara- 
tions. The  thread  on  the  nucleus  in  fig.  66  shows  a  row  of 
chromatin  granules  and  soon  becomes  segmented  into  twelve 
chromosomes.  If  the  stain  is  too  dense,  the  thread  will  probably 
appear  smooth  throughout  all  these  stages,  but  if  the  staining  is 
successful,  the  granules  are 
sharply  stained  by  the  gen- 
tian violet,  while  the  linin 
thread  is  stained  lightly  or 
not  at  all.  In  successful 
staining  with  cyanin  and 
erythrosin  the  granules 
stain  blue  and  the  linin  red. 
Stages  like  figs.  6y  and  68 
are  easily  stained,  and  the 
preparations  are  exception- 
a  1 1  y  beautiful.  Stain  for 
twenty-four  hours  in  safra- 
nin  (the  solution  in  50  per 
cent,  alcohol  is  very  good), 
and  then  rinse  in  50  per  cent,  alcohol  until  the  red  color 
disappears  from  the  spindle,  but  remains  bright  in  the  chro- 
mosomes and  nucleoli ;  stain  in  gentian  violet  four  to  eight 
minutes;  rinse  in  water  about  thirty  seconds ;  stain  in  aqueous 
orange  G  fifteen  to  thirty  seconds ;  transfer  to  absolute  alcohol, 
and  move  the  slide  gently  back  and  forth  in  order  to  dehydrate 
as  rapidly  as  possible  (three  to  six  seconds  will  usually  be  long 
enough);  the  slide  must  be  taken  from  the  absolute  alcohol 
while  the  gentian  violet  is  still  coming  out  in  streams  ;  treat  with 
clove  oil  ten  to  thirty  seconds,  and  then  drain  off  the  clove  oil 
and  add  a  few  drops  of  cedar  oil,  since  the  gentian  violet  fades  if 
much  clove  oil  is  left  in  the  preparation.     If  the  cedar  oil  is 


Fig.  65.    Lilium  philadelphicum.     X  710. 

Apex  of  nucellus  contains  a  large  archesporial  cell  with  a 
large  nucleus.  In  Lilium  this  archesporial  cell  becomes 
the  embryo-sac  directly  without  cutting  off  any  tapetal 
cell  or  dividing  into  potential  megaspores.  Fixed  in 
chromo-acetic  acid  and  stained  in  Delafield's  haematoxylin. 


34 


Methods  in  Plant  Histology 


transparent  and  has  a  strong  odor,  do  not  use  it,  but  remove  as 
much  of  the  clove  oil  as  possible.  Mount  directly  in  balsam. 
Before  transferring  to  absolute  alcohol,  a  single  dip  in  95  per 
cent,  alcohol  may  do  no  damage  and  is  a  matter  of  economy, 
since  it  avoids  carrying  so  much  water  into  the  absolute  alcohol. 

When  the 
staining  is 
properly  done, 
the  chromo- 
somes will 
show  a  bright 
red  color  and 
the  spindle  a 
brilliantviolet. 
If  the  action 
of  the  gentian 
violet  be  too 
prolonged  o  r 
poorly  ex- 
tracted, the 
chromosomes 
will  appear 
violet  or  red- 
dish violet, 
and  such  de- 
tails as  the  re- 
lation of  spin- 
d  1  e  fibers  t  o 
chromosomes 
will  be  ob- 
scured. Stages 
shown  in  figs. 

6g  and  70  may  be  found  in  the  same  ribbons  with  stages  like^^.y. 
d/  and  68,  and  are  well  differentiated  by  the  same  treatment. 

Later  stages  up  to   fertilization  and  the  first  divisions  of  the 
embryo  may   be   fixed  and    stained    as    already   described,  but 


Fig.  66.     Lilium  philadelphicum.     X  710. 

Nucellus  with  megaspore.  The  chromatin  thread  in  the  nucleus  is  very 
distinct.  Fixed  in  chromo-acetic  acid  and  stained  in  safranin-gentian  violet- 
orange. 


Spermatophytes 


135 


cyanin  and  erythrosin  gives  a  particularly  brilliant  effect, 
especially  after  Carney's  fluid.  At  the  stage  shown  in  fig.  yi, 
the  male  nucleus  takes  the  cyanin,  and  the  oosphere  nucleus  the 
erythrosin,   although  at  a  slightly  later  stage  they  stain  alike. 


Fig.  67.  Lilium  philadelphicum.     X  710.  Fig.  6S. 

First  division  of  the  nucleus  of  the  megaspore.  _  (The  same  nucleus  the  earlier  stages  of  which  are 
shown  in  figs.  65  and  66.)  Fixed  in  chromo-acetic  acid,  and  stained  in  safranin- gentian  violet-orange. 
Fifteen  microns. 

For  the  later  stages  in  the  development  of  the  embryo,  Delafield's 
haematoxylin  is  a  far  better  stain. 

Many  of  the  Compositae,  like  Aster,  Taraxacum,  Senecio,  and 
Silphmm,  are  excellent  for  a  study  of  the  mature  sac  and  the 
formation  of  the  embryo.  The  whole  head  may  be  cut  if  trimmed 
as  indicated  '\n  fig.  64,  but  for  the  embryo-sac  at  the  fertilization 
period,  and  also  for  the  development  of  the  embryo,  it  is  worth 
while  to  resort  to  the  tedious  process  of  dissecting  the  ovules 


136 


Methods  in  Plant  Histology 


out  from   the  ovaries.     Stages  like  those  shown  in  figs.  6^-yo 
are  rather  unsatisfactory  in  Composites. 

Many    of   the    Ranunculaceae   are    easily   studied.     Anemone 
patens,  var.  Nuttalliana,  has  a  very  beautiful  embryo-sac,  the  tgg, 


Fig.  69.     Lilium  philadelphicum.     X  710. 

Megaspore  (embryo-sac)  containing  two 
daughter-nuclei  resulting  from  the  first  division  of 
the  nucleus  of  the  megaspore.  A  portion  of  the 
spindle  still  remains  between  the  two  nuclei. 
Fixed  in  chromo-acetic  acid,  and  stained  in  safra- 
nin-gentian  violet-orange.     Fifteen  microns  thick. 


Fig.  70.     Li! 


philadelphicum.     X  710. 


Later  stage  in  the  development  of  the  embryo- 
sac.  Each  of  the  two  nuclei  shown  in  fig.  69  has 
divided.  The  nuclei  are  much  smaller  than  those 
in  fig.  69.  Fixed  in  chromo-acetic  acid,  and 
stained  in  safranin-gentian  violet-orange.  Fifteen 
microns  thick. 


synergids,  endosperm  nucleus,  and  antipodals  being  rather  large 
and  sharply  defined.  Hepatica,  Caltha,  and  some  species  of 
Ra?iunculus  are  exceptionally  good. 

Development  of  the  Embryo. —  The  common  shepherd's  purse 
{Capsella  btirsa-pastoris)  is  a  favorable  form  for  a  study  of  the 
development  of  a  dicotyl  embryo.    The  stages  shown  in  fig.  y2, 


Spermatophytes 


137 


A-F,  will  be  found  in  pods  which  are  about  one-eighth  of  an 
inch  long.  These  may  be  put  directly  into  the  fixing  agent,  but 
stages  like  G  and  //are  found  in  pods  about  three-sixteenths  of 
an  inch  long,  and  such  stages  will  be  more  readily  fixed,  infil- 
trated, and  cut  if  the  pods  are  trimmed,  as  shown  '\i\fig.  64,  B, 
before  putting  them  into  the  fixing 
agent.  Cut  sections  parallel  to  the 
flat  face  of  the  pod.  Delafield's 
hsematoxylin,  without  any  contrast 
stain,  gives  the  best  results  which 
we  have  secured. 

For  tracing  the  development  of 
a  monocotyl  embryo  Sagittaria  vari- 
abilis can  be  recommended.  Mate- 
rial is  abundant,  sections  are  easily 
cut,  except  in  the  latest  stages,  and 
it  is  not  difficult  to  get  a  complete 
series.  Alisma  plaiitago,  which  is 
commonly  figured  in  text-books,  is 
extremely  hard  to  cut,  especially  in 
later  stages. 

Leaves. — Where  only  a  rapid  ex- 
amination is  to  be  made,  free-hand 
sections  may  be  made  in  great  num- 
bers by  using  the  method  employed 
for  pine  needles.  It  is  easy  to  get 
good  sections  of   leaves  which  can 

,                             .                         -_,..,.-_  Embryo-sac  at  time  of  fertilization.     A, 

be   gotten   mtO   paraffin,  but   it  is  dlffl-  the  three  amipodals.    £•,  protoplasm  of  the 

sac.      e,   polar    nuclei    fusing   to    form   the 

cult    to    get    tender,   succulent   leaves  endosperm    nucleus  ;    the   male   nucleus   is 

®  about    to     fuse    with    the    nucleus    of    the 

into  paraffin  without  distortion.     Such  oo^phere.       z     the    inner    integument.    // 

J^  the  pollen   tube.     Y  ixed   in   Carnoy  s  tluid 

leaves  may  be  cut  in  celloidin.     For  fe" n SnVtVcr' (iTat^'f CyS": 

a  study  of  the  StOmata,   strip  a  piece    Pedia  of  American  Horticulture.) 

of  epidermis  from  the  leaf,  fix  it,  stain  in  Delafield's  haematoxylin 
and  erythrosin,  pass  it  gradually  through  the  alcohols,  clear  in 
xylol,  and  mount  in  balsam.  Lily,  tulip,  hyacinth,  and  begonia 
may  be  suggested  as  favorable  forms.     Epidermis  from  the  leaf 


Lilium  philadelphicum.     X  335. 


138 


Methods  in  Plant  Histology 


of  the  common  Sedum  pupurasce?is  will  usually  show  stomata  in 
all  stages  of  development. 

Stems  and  Roots. — The  earlier  stages  in  the  development  of 
vascular  bundles  in  stems  and   roots  are  well   shown    in   parafifin 


Fig.  72.     Capsella  bursa-pastoris,     X  400. 

^ ,  first  division  in  the  embryo  cell.  ^,  quadrants.  C,  octants,  i?,  the  dermatogen  has  been  cut 
off.  There  are  eight  cells  in  the  suspensor,  the  lower  cell  being  very  large  and  vesicular.  E.  differ- 
entiation in  plerome  and  periblem.  The  plerome  cells  are  shaded.  F,  the  periblem  of  the  root  is  com- 
pleted at  the  expense  of  the  upper  cell  of  the  suspensor.  G,  the  mitotic  figure  in  the  suspensor  cell  indi- 
cates that  the  upper  suspensor  cell  by  a  second  contribution  is  about  to  complete  the  dermatogen  of  the 
root.  H,  plerome  (shaded),  periblem,  dermatogen  (shaded),  and  first  layer  of  the  root  cap.  Fixed  in 
chromo-acetic  acid  and  stained  in  Delafield's  hsematoxylin.     Ten  microns  thick. 

sections  of  young  seedlings.     The  common  bean  is  a  favorable 
form,  and  it  is  easy  to  get  material. 

Most  herbaceous  stems  and  roots,  and  also  the  younger 
woody  stems  and  roots,  give  the  best  results  when  cut  in  celloidin, 
as  already  described  for  Pinus.  Rumex  crispus  and  Ranunculus 
repens  can  be  recommended  for  a  study  of  the  vascular  bun- 
dles.    The  cambium  is  very  sharply  brought  out  by  Delafield's 


Spermatophytes 


139 


haematoxylin.  Petioles  or  leaf  blades  of  A^zz/Zz^r  imbedded  in  cel- 
loidin,  and  stained  in  safranin  and  Delafield's  haematoxylin,  yield 
extremely  beautiful  preparations,  the  sclerotic  cells  taking  a 
brilliant  red  and  the  cellulose  a  rich  purple.  The  sections  should 
be  20-30  /li  thick. 

Stems  or  petioles  of  the  squash  or  pumpkin  are  to  be  pre- 
ferred for  demonstrating  sieve  tubes  and  companion  cells.  For 
the  more  minute  de- 
tails of  the  sieve  plate 
it  is  best  to  cut  out 
small  piecesaboutone- 
fourth  of  an  inch  long 
and  one-eighth  of  an 
inch  square  containing 
the  vascular  bundle. 
These  pieces  can  be 
imbedded  in  paraffin. 

For  demonstrating 
the  phellogen  and  the 
tissue  developed  from 
it,  stems  of  Geranium 
or  Coleus  about  one- 
fourth  of  an  inch  in 
diameter  or  seedlings 
of  Xanthium  canadense 
about. three-sixteenths 
of  an  inch  in  diameter 
can  be  recommended. 
They  can  be  cut  in  paraffin,  but  satisfactory  results  are  more 
uniformly  obtained  from  celloidin  sections. 

The  stem  of  Indian  corn,  imbedded  in  celloidin  and  stained 
in  safranin  and  Delafield's  haematoxylin,  affords  a  good  study  of 
monocotyl  stem  anatomy. 

It  must  not  be  forgotten  that  root  tips,  besides  showing  ana- 
tomical structure,  furnish  ever-ready  material  for  the  study  of 
karyokinesis.     An  onion  thrown  into  a  pan  of  water  will  soon 


Fig.  73.     Sparganium   eurycarpum.     X  53. 

Tranverse  section  of  a  root.     Delafield's  haematoxyli 
and  acid  fuchsin.     Five  microns. 


140  Methods  in  Plant  Histology 

send  out  numerous  roots.  About  one-fourth  of  an  inch  should 
be  cut  off  from  the  tip  of  the  root.  Fix  and  stain  as  directed 
for  the  mitotic  figures  in  Lilium.  While  the  onion  is  very  avail- 
able material,  the  figures  are  not  as  satisfactory  as  those  to  be 
obtained  in  the  root  tips  of  Tradescantia  virginica,  Iris  versicolor, 
Podyphyllum.  peltatum,  Arismma  triphyllum,  Cypripedium  pubescens, 
and  many  others.  Transverse  sections  of  young  roots  often  show 
a  remarkably  regular  arrangement  of  the  cells,  as  can  be  seen  in 

fig'  73- 

We  are  painfully  aware  that  the  directions  which  have  been 
given  in  this  series  of  articles  are  very  incomplete,  but  it  is  hoped 
that  they  will  enable  the  student  to  devise  for  himself  such 
methods  as  particular  cases  may  demand. 


CHAPTER  XX. 
LABELING  AND  CATALOGUING  PREPARATIONS. 

THE    LABEL. 

The  labels  shown  in  fig.  62,  on  p.  126,  show  as  much  as 
will  generally  be  found  desirable.  The  date  of  the  collection 
of  the  material  is  often  needed  in  addition.  The  date  of  making 
the  preparation  is  of  no  value  unless  the  student  is  testing  the 
permanence  of  stains  or  something  of  that  sort.  It  is  hardly 
worth  while  to  write  upon  the  label  the  names  of  the  stains  used, 
for  the  student  will  soon  learn  to  recognize  the  principal  stains. 
We  should  say  that  the  first  tKmg  to  write  upon  a  label  is  the 
genus  and  species  of  the  plant ;  the  next  thing  would  be  the 
name  of  the  organ  or  tissue,  and  then  might  be  added  the  date 
of  collection :  e.  g.,  Marchantia  polymorpha,  young  archegonia, 
April  10,  1 90 1.  A  hasty  sketch  on  the  label  will  often  indicate 
any  exceptionally  interesting  feature  in  the  preparation.  To 
facilitate  finding  such  a  feature,  it  is  a  good  plan  to  mark  the 
particular  Section  or  sections  with  ink,  the  marking  being 
always  on  the  underside  of  the  slide  so  as  not  to  cause  any 
inconvenience  if  an  immersion  lens  should  be  used. 

CATALOGUING  PREPARATIONS. 

As  a  collection  grows,  the  student  will  need  some  device  for 
readily  locating  any  particular  preparation.  Some  have  their 
slides  numbered  and  catalogued,  but  all  devices  of  this  sort  are 
too  cumbrous  and  slow  for  the  practical  worker  in  the  labora- 
tory. After  several  years'  experience  with  a  collection  which 
now  numbers  about  seven  thousand  preparations,  the  following 
method  can  be  confidently  recommended : 

Four  wooden  slide  boxes  of  the  usual  type  will  do  for  a 
beginning ;  they  should  be  labeled :  Thallophyta,  Bryophyta, 
Pteridophyta,  and  Spermatophyta.     As  the  collection  grows 

141 


142 


Methods  i?t  Plant  Histology 


and  new  boxes  are  needed,  the  classification  can  be  made  more 
definite  ;  e.g.,  there  should  be  a  box  labeled  Bryophytes  HepaticcB 
and  one  labeled  Bryophytes  Musci.  As  the  liverwort  collection 
grows,  three  boxes  will  be  necessary,  and  should  be  labeled 
Bryophytes  Hepaticce  Marchantiales,  Bryophytes  HepaticcE  Jun- 
germanniales,  and  Bryophytes //"(f/^/zV^Anthocerotales.  It  will 
readily  be  seen  that  the  process  can  be  continued  almost  indefi- 
nitely, and  that  new  slides  may  be  at  any  time  dropped  into  their 
proper  places.  A  rather  complete  label  gradually  built  up  in 
this  way  is  shown  '\n  fig.  7^/ 


BRYOPHYTES 

HEPATIC^ 

Jungermanniales 

Porella    platyphyllum 

Archegonia 

CHAPTER  XXI. 

A  CLASS  LIST  OF  PREPARATIONS. 

Where  a  regular  course  in  histology  is  conducted,  it  is  a  good 
plan  to  give  each  student  at  the  outset  a  complete  list  of  the 
preparations  which  he  is  expected  to  make.  In  a  three-months' 
course  a  fairly  representative  collection  of  preparations  can  be 
made.  The  availability  of  material  determines  what  a  list  shall 
be.  The  following  list  was  recently  used  by  one  of  the  writer's 
classes : 

LIST  OF   PREPARATIONS. 

THALLOPHYTES. 

ALG^. 

CYANOPHYCE^. 

1.  Wasserbliithe. — The  principal  forms  in  this  material  are : 

(a)  Coelosphariuni   Kutzingianum.  —  Colonies    in    the    form    of   hollow 

spheres. 
(^)   Anabcena  gigantea. —  Filaments  straight.    Preparations  should  show 

vegetative  cells,  heterocysts,  hormogonia,  and  spores. 
(<r)   Anabcena  flos-aquce. —  Filaments   curved.      Stain   on    the  slide    and 

mount  in  balsam. 

2.  Nostoc. — Eosin  or  erythrosin.     Glycerine. 

3.  Oscillaria. —  Put  living  material  into  10  per  cent,  glycerine  and  allow  it 
to  concentrate. 

4.  Glo2otrichia. —  Eosin,  erythrosin,  or  iron  alum-haematoxylin.  Glycerine. 
Should  show  heterocysts,  spores,  and  vegetative  filaments.     . 

5.  Tolypothrix. — Treat  like  Oscillaria  or  Gloeotrichia.  Should  show  hetero- 
cysts, hormogonia,  and  false  branching. 

CHLOROPHYCEiE. 

6.  Volvox. —  Stain  some  in  eosin  or  erythrosin  and  some  in  very  dilute 
Delafield's  haematoxylin.  Mount  in  glycerine  in  a  cell  deep  enough  to 
prevent  crushing.  Try  to  have  antheridia,  oogonia,  and  young  colonies 
in  each  mount. 

7.  Vaucheria. —  Eosin,  erythrosin,  or  iron  alum-hsematoxylin.  Glycerine. 
Should  show  antheridia,  oogonia,  zoospores. 

143 


144  Methods  in  Plant  Histology 

8.  Hydrodictyon. —  Iron  alum-hsematoxylin  or  dilute  Delafield's  haematoxy- 
lin.     Glycerine.     Look  for  young  nets  inside  the  older  segments. 

9.  Spirogyra. —  Iron  alum-hsematoxylin.  A  counter-stain  with  eosin  is  a 
great  improvement,  if  successful.  Glycerine.  Should  show  chroma- 
tophores,  pyrenoids,  and  nucleus.  Eosin,  erythrosin,  or  dilute  Delafield's 
haematoxylin  will  be  better  for  conjugating  material. 

I  o.  Zygnema. —  Same  treatment  as  for  Spirogyra  to  bring  out  chromatophores, 
pyrenoids,  and  nucleus.  Use  iron  alum-haematoxylin  for  reproductive 
stages. 

1 1.  Diatoms. —  Living  and  fossil  forms.     Balsam. 

12.  Oedogonium. —  Use  iron  alum-haematoxylin  for  oogonia,  antheridia,  and 
zoospores.  Eosin  or  erythrosin  for  caps  and  other  vegetative  structures. 
Glycerine. 

13.  Chara. —  Oogonia  and  antheridia  may  be  mounted  whole  in  a  cell  deep 
enough  to  prevent  crushing.  Glycerine  jelly.  Paraffin  sections  of 
oogonia,  antheridia,  and  apical  cell. 

PHiEOPHYCEiE. 

14.  Ectocarpus. —  Stain  some  in  eosin  and  some  in  dilute  Delafield's  haema- 
toxylin. Glycerine.  Each  mount  should  show  both  unilocular  and  mul- 
tilocular  sporangia. 

15.  Fucus  vesiculosus. —  Antheridial  conceptacle  with  paraphyses  and  anthe- 
ridia ;  oogonial  conceptacle  with  oogonia.  Delafield's  haematoxylin  for 
paraffin  sections.     Borax  carmine  for  teased  preparations. 

RHODOPHYCEiE. 

J  6.    Batrachospermuvi    (or    Nemalion).  —  Mayer's    hasm-alum.      Glycerine. 

Should  show  trichogyne,  carpogonium,  and  cystocarp. 
J/.    Polysiphonia  fibrillosa. —  Iron  alum-haematoxylin  or  eosin.      Glycerine. 
Vegetative  structure,  tetraspores,  cystocarps  with  carpospores,  antheridia. 
FUNGI. 
MYXOMYCETES. 

J  8.     Trichia  varia. —  Paraffin  sections,  5/*.     Safranin-gentian  violet-orange. 

SCHIZOMYCETES. 

1 9.  Bacteria. —  Coccus,  Bacillus,  and  Spirillum  forms.  Stain  on  cover-glass 
or  slide. 

20.  Bacillus  anthracis. —  In  liver  of  mouse.  Paraffin  sections,  5  /*.  Stain 
in  gentian  violet,  Gram's  method. 

PHYCOMYCaW"ES. 

21.  Mucor  stolonifer. —  Stain  young  sporangia  in  eosin  or  dilute  Delafield's 
haematoxylin.  Zygosporic  material  may  be  mounted  without  staining  or 
after  a  very  light  staining  in  dilute  Delafield's  haematoxylin.     Glycerine. 


Class  List  of  Preparations  145 

22.  Cystopus  candidus  on  Cakile  americana. —  Select  white  blisters  which 
have  not  yet  broken  open.     Paraffin,  5  m-    Safranin-gentian  violet-orange. 

23.  Cystopus  bliti  on  Amarantus  retroflexus. —  Cut  out  small  portions  of 
leaves  in  which  the  oogonia  can  be  seen  in  abundance.     Paraffin,  5  ^x.. 

ASCOMYCETES. 

24.  Eurotium. —  Eosin  or  erythrosin.     Glycerine. 

25.  Pencillium. —  Eosin  or  erythrosin.     Glycerine. 

26.  Erysiphe  cotnmune  on  Polygonum  aviculare. —  Strip  the  fungus  from  the 
leaf.     Paraffin,  5  /*.     Safranin-gentian  violet-orange. 

27.  Uncinula  necator  on  Ampelopsis  quinquefolia.  —  Eosin  or  erythrosin. 
Mount  whole  and  break  the  perithecia  under  the  cover.  Glycerine  or 
balsam. 

28.  Xylaria. —  Paraffin  sections  of  younger  stages.  Delafield's  haematoxj'lin 
and  erythrosin.  Be  sure  that  some  section  in  each  mount  shows  the 
opening  of  a  perithecium. 

29.  Peziza. —  Paraffin  sections  of  young  apothecia,  5  m  or  less ;  sections  of 
older  apothecia,  lO/t  or  15  /it.     Safranin-gentian  violet-orange. 

iECIDIOMYCETES. 

30.  Pticcifiia  graminis. —  ^cidium  stage  on  barberry  leaf.  Paraffin.  Cyanin 
and  erythrosin.  Uredospore  and  teleutospore  stage  in  celloidin  or  paraffin, 

BASIDIOMYCETES. 

3 1 .  Coprittus  comatus. —  Paraffin.  Transverse  sections  of  gills  showing  trama, 
paraphyses,  basidia,  and  spores.  To  show  the  basidium  with  four  spores, 
the  sections  should  be  15  m  thick.  For  development  of  the  spores,  cut 
5  M  or  less.  Safranin-gentian  violet-orange.  Boletus,  Hydnuni,  and 
Polyporus  are  treated  in  the  same  manner. 

LICHENS. 

32.  Parmelia  Borreri. —  Paraffin,  5/^.    Cyanin  and  erythrosin. 

BRYOPHYTES. 
HEPATIC^. 

33.  Ricciocarpus  nutans. —  Paraffin,  iom  or  15^;  Delafield's  haematoxylin. 
Archegonia,  antheridia,  and  sporophytes  imbedded  in  the  gametophyte. 

34.  Marchantia  polyniorpha. —  Paraffin,  lo/i.  Archegonia,  antheridia,  and 
sporophytes. 

35.  Pellia  epiphylla. —  Paraffin,  lo/x.  Longitudinal  sections  of  sporophyte 
attached  to  gametophyte.     Delafield's  haematoxylin  and  erythrosin. 

36.  Porella  platyphyllufu. —  Paraffin,  10 /x.  Delafield's  haematoxylin.  Arche- 
gonia, antheridia,  sporophyte,  and  apical  cell. 

37.  Anthoceros  Icevis. —  Paraffin,  5  or  10 ix.  Longitudinal  and  transverse 
sections  of  sporophyte.     Safranin-gentian  violet-orange. 


146  Methods  i?i  Plant  Histology 

MUSCI. 

38.  Sphagnum. —  Leaf  buds.  Safranin  and  Delafield's  haematoxylin.  Tease 
and  mount  in  balsam. 

39.  Sphagnum. —  Capsule.  Parafifin.  Delafield's  haematoxylin  and  erythro- 
sin. 

40.  Bryum  proliferum. —  Parafifin.  Antheridia,  lO/tt ;  archegonia,  15  to20ju; 
capsule,  10  At. 

41.  Funaria  hygrometrica. —  Paraffin.  Longitudinal  and  transverse  sections 
of  young  capsules.     Delafield's  haematoxylin. 

42.  Funaria  hygrometrica  or  any  favorable  form.  Protonema.  Place  the 
well-cleaned  material  directly  into  10  per  cent,  glycerine  and  allow  it  to 
concentrate. 

PTERIDOPHYTES. 

FILICALES. 

43.  Botrychium  virginianum. —  Paraffin.  Stain  rhizome,  stipe,  and  root  in 
safranin  and  Delafield's  haematoxylin.  Stain  sporangia  in  Delafield's 
haematoxylin. 

44.  Isoetes  echinosPora. —  Transverse  section  of  stem.  Celloidin.  Safranin 
and  Delafield's  haematoxylin. 

45.  Isoetes  echinospora. —  Paraffin.  Longitudinal  sections  of  microsporangia 
and  megasporangia.     Safranin-gentian  violet-orange. 

46.  Pteris  aquilina. —  Free-hand  sections.  Safranin  and  Delafield's  haematox- 
ylin, or  iodine  green  and  fuchsin.     Balsam. 

47.  Pteris  cretica.  —  Paraffin.  Transverse  sections  of  leaves  with  sporangia. 
Delafield's  haematoxylin  and  erythrosinor  safranin-gentian  violet-orange. 

48.  Adiantum  cuneatum.—  Prothallia  mounted  whole  in  glycerine.  Paraffin 
sections  of  antheridia  and  archegonia. 

49.  Marsilea  quadrifolia. —  Parafifin.  Longitudinal  and  transverse  sections 
of  sori.     Safranin-gentian  violet-orange. 

50.  Azolla  carolinense. —  Parafifin.  Vertical  sections  of  the  whole  plant 
showing  micro-  and  megasporangia. 

EQUISETALES. 

51.  Equisetum  arvense. —  Prothallia  in  glycerine.  Stem  tips  in  parafifin. 
Transverse  section  of  stem  in  celloidin. 

LYCOPODIALES. 

52.  Lycopodium  lucidulum. —  Transverse  section  of  stem.  Free-hand  or  in 
celloidin.     Safranin  and  Delafield's  haematoxylin. 

53.  Lycopodium  inundatum. —  Paraffin.     Longitudinal   sections  of  strobilus. 

54.  Seiaginella. —  Paraffin.  Longitudinal  sections  of  rather  mature  strobili. 
Cyanin  and  erythrosin. 


Class  List  of  Preparations  147 

SPERMATOPHYTES. 

GYMNOSPERMS. 

55.  Pinus  Laricio. — Transverse  section  of  needles  and  young  stem.  Free- 
hand or  cefloidin.     Safranin  and  Delafield's  hasmatoxylin. 

56.  Pinus  strobus. —  Free-hand  sections  of  well-seasoned  wood.  Iodine 
green  and  fuchsin,  or  safranin  and  Delafield's  haematoxylin. 

57.  Pinus  Laricio. —  Paraffin.  Ovule  with  archegonia.  Safranin-gentian 
violet-orange. 

58.  Pinus  sylvestris  or  P.  Laricio. — -Paraffin.     Embryos. 

59.  Pinus  Laricio. —  Paraffin.  Longitudinal  section  of  mature  staminate 
strobilus.     Safranin-gentian  violet-orange. 

ANGIOSPERMS. 
MONOCOTYLS. 

60.  Lilium  philadelphicum. —  Paraffin,  10  fj..  Transverse  sections  of  ovary; 
transverse  sections  of  anthers. 

61.  Tradescantia  virgittica. —  Paraffin,  5  and  i  o  joc.  Longitudinal  sections  of 
root.     Stain  for  mitosis. 

62.  Allium. —  Paraffin.  Transverse  sections  of  older  roots  for  vascular 
system. 

63.  Liliufn  longiflorum. —  Strip  epidermis  from  leaf  and  stain  for  stomata. 
Cut  sections  of  leaf  in  paraffin,  10  to  20  /x. 

64.  Zea  mais. —  Celloidin.     Longitudinal  and  transverse  sections  of  stem. 

DICOTYLS. 

65.  Nuphar  advena. —  Celloidin.  Section  of  leaf  1 5  to  40  yn  thick.  Safranin 
and  Delafield's  haematoxylin. 

66.  Capsella  bursa-pastoris. —  Paraffin.  Floral  development,  5 /*.  Embryos, 
5  to  lo/x.  Stain  both  in  Delafield's  haematoxylin  without  any  contrast 
stain. 

67.  Taraxacum  officinale. —  Paraffin.  Floral  development,  5 /u.  Embryo- 
sac,  10  to  15  \t.. 

68.  Anemone  patens. —  Paraffin.     Embryo-sac. 

69.  Smilax  herbacea. —  Celloidin.  Transverse  section  of  root.  Safranin 
and  Delafield's  haematoxylin. 

70.  Xanthium  canadense. —  Celloidin.  Transverse  section  of  stem  of  seed- 
ling to  show  cambium  and  phellogen. 

71.  Tilia  atnericana. —  Celloidin  or  free-hand.  Transverse  sections  of  small 
stems  one-eighth  to  one-fourth  of  an  inch  in  diameter.  SafrAnin  and 
Delafield's  haematoxylin. 

In  making  the  mounts  the  order  indicated  in  the  list  should 
not  be  followed.     Begin  with  free-hand  sections,  then  study  the 


148  Methods  in  Plant  Histology 

glycerine  method,  and  then  devote  a  large. portion  of  the  time  to 
the  paraffin  method.     Let  the  celloidin  method  come  last. 

It  is  neither  possible  nor  desirable  that  each  student  should 
in  every  case  go  through  all  the  processes  from  collecting  mate- 
rial to  labeling.  Some  of  the  material  may  be  in  70  per  cent, 
alcohol,  some  in  formalin,  some  in  glycerine,  and  some  in  paraffin. 
One  student  may  imbed  in  paraffin  enough  of  the  A?iemone  for 
the  whole  class,  another  may  imbed  the  Lilium  stamens,  and  by 
such  a  division  of  labor  a  great  variety  of  preparations  may  be 
secured  without  a  corresponding  demand  upon  the  time  of  the 
individual. 


CHAPTER  XXII. 

FORMULA  FOR  REAGENTS. 
FIXING  AGENTS. 

Carnoy's  Fluid. — 

Absolute  alcohol,  6  parts. 
Chloroform,  3  parts. 
Glacial  acetic  acid,  i  part. 
Strong  Chromo-Acetic  Solution. — 
Chromic  acid,  i  g. 
Glacial  acetic  acid,  i  cc. 
Water,  98  cc. 

Weak  Chromo-Acetic  Solution  (Schaffner's) . — 

Chromic  acid,  0.3  g. 
Glacial  acetic  acid,  0.7  cc. 
Water.  99  cc. 

Medium  Chromo-Acetic  Solution. — 

Chromic  acid,  0.7  g. 
Glacial  acetic  acid,  0.5  cc. 
Water,  100  cc. 
Flemming's  Fluid  (weaker  solution). — 
(  I  per  cent,  chromic  acid,  25  cc. 
A  ■<  I  per  cent,  acetic  acid,  10  cc, 

(  Water,  55  cc. 
B.    I  per  cent,  osmic  acid,  55  cc. 

Keep  the  mixture  A  made  up,  and  add  B  as  the  reagent  is 
needed  for  use,  since  it  does  not  keep  well. 

Flemming's  Fluid  (stronger  solution). — 

1  per  cent,  chromic  acid,  45  cc. 

2  per  cent,  osmic  acid,  12  cc. 
Glacial  acetic  acid,  3  cc. 

MerkePs  Fluid. — 

1.4  per  cent,  solution  of  chromic  acid,  25  cc. 
1.4  per  cent,  solution  of  platinic  chloride,  25  cc. 

Hermann's  Fluid. — 

1  per  cent,  platinic  chloride,  15  parts. 
Glacial  acetic  acid,  i  part. 

2  per  cent,  osmic  aid,  4  or  2  parts. 

149 


150  Methods  m  Plant  Histology 

Picric  Acid. — 

Picric  acid,  i  g. 

Water,  or  70  per  cent,  alcohol,  100  cc. 

Corrosive  Sublimate. — 

Bichloride  of  mercury,  4  g. 

Glacial  acetic  acid,  a  cc. 

Water,  or  70  per  cent,  alcohol,  100  cc. 

Formalin  (weaker  solution). — 
Formalin,  2  cc. 
Water,  98  cc. 

Formalin  (stronger  solution). — 
Formalin,  4  cc. 
Water,  100  cc. 

Osmic  Acid. — 

Osmic  acid,  i  cc. 
Distilled  water,  100  cc. 

The  bottle  in  which  the  solution  is  to  be  kept,  and  also  the 
glass  tube  in  which  the  acid  is  sold,  must  be  thoroughly  cleaned. 
Break  off  the  end  of  the  tube,  and  drop  both  tube  and  acid  into 
the  distilled  water. 

STAINS. 

Delafield's  Haematoxylin. — "To  100  cc.  of  a  saturated  solution 
of  ammonia  alum  add,  drop  by  drop,  a  solution  of  l  g.  of  haema- 
toxylin dissolved  in  6  cc.  of  absolute  alcohol.  Expose  to  air 
and  light  for  one  week.  Filter.  Add  25  cc.  of  glycerine  and 
25  cc.  of  methyl  alcohol.  Allow  to  stand  until  the  color  is  suf- 
ficiently dark.  Filter  and  keep  in  a  tightly  stoppered  bottle." 
(Stirling  and  Lee.) 

The  solution  should  stand  for  at  least  two  months  before  it 
is  ready  for  using. 

Erlich's  Haematoxylin. — 

Distilled  water,  50  cc. 
Absolute  alcohol,  50  cc. 
Glycerine,  50  cc. 
Glacial  acetic  acid,  5  cc. 
Haematoxylin,  i  g. 
Alum  in  excess. 
Keep  it  in  a  dark  place  until  the  color  becomes  a  deep  red. 
If  well  stoppered,  it  will  keep  indefinitely. 


FormulcB  for  Reagents  151 

Boehmer's  Haematoxylin. — 

Hsematoxylin,  i  g. 
Absolute  alcohol,  12  cc. 
Alum,  I  g. 
Distilled  water,  240  cc. 

The  solution  A  must  ripen  for  two  months.  When  wanted 
for  use,  add  about  10  drops  of  A  to  10  cc.  of  B.  Stain  ten  to 
twenty  minutes.     Wash  in  water  and  proceed  as  usual. 

Mayer's  Haem-Alum.  —  Haematoxylin,  i  g.,  dissolved  with 
heat  in  5a  cc.  of  95  per  cent,  alcohol  and  added  to  a  solution  of 
50  g.  of  alum  in  a  liter  of  distilled  water.  Allow  the  mixture  to 
cool  and  settle ;  filter  ;  add  a  crystal  of  thymol  to  preserve  from 
mold.    (Lee.) 

It  is  ready  for  use  as  soon  as  made  up.  Unless  attacked  by 
mold,  it  keeps  indefinitely. 

Haidenhain's  Iron  Alum-Haematoxylin. — This  stain  was  intro- 
duced by  Haidenhain  in  1892  and  has  gained  a  well-deserved 
popularity  with  those  engaged  in  cytological  work.  Two  solu- 
tions are  used,  and  they  are  never  mixed  : 

A.  One  and  one-half  to  4  per  cent,  aqueous  solution  of 
ammonia  sulphate  of  iron.  (At  present  we  use  a  3  per  cent, 
solution.) 

B.  One-half  per  cent,  aqueous  solution  of  haematoxylin. 
Greenacher's  Borax  Carmine. — 

Carmine,  3  g. 

Borax,  4  g. 

Distilled  water,  xoo  cc. 

Dissolve  the  borax  in  water  and  add  the  carmine,  which  is 
quickly  dissolved  with  the  aid  of  gentle  heat.  Add  lOO  cc.  of 
70  per  cent,  alcohol  and  filter.      (Stirling.) 

Alum  Carmine. — A  4  per  cent,  aqueous  solution  of  ammonia 
alum  is  boiled  twenty  minutes  with  i  per  cent,  of  powdered  car- 
mine.    Filter  after  it  cools.      (Lee.) 

Alum  Cochineal. — 

Powdered  cochineal,  50  g. 

Alum,  5  g. 

Distilled  water,  500  cc. 


152  Methods  i?i  Pla?it  Histology 

Dissolve  the  alum  in  water,  add  the  cochineal,  and  boil ; 
evaporate  down  to  two-thirds  of  the  original  volume,  and  filter. 
Add  a  few  drops  of  carbolic  acid  to  prevent  mold.      (Stirling.) 

Picro-Carmine. — 

Picro-carmine  (picro-carminate  of  ammonia),  i  g. 
Water,  100  cc. 

Eosin. — 

Eosin,  1  g. 

Water,  or  70  per  cent,  alcohol,  100  cc. 

General  Formula  for  Anilins. — Make  a  3  per  cent,  solution  of 
anilin  oil  in  distilled  water  ;  shake  well  and  frequently  for  a  day; 
add  enough  alcohol  to  make  the  whole  mixture  about  20  per 
cent,  alcohol ;  add  i  g.  of  cyanin,  erythrosin,  safranin,  gentian 
violet,  etc.,  to  each  100  cc.  of  this  solution. 

Iodine  Green. — 

Iodine  green,  i  g. 

70  per  cent,  alcohol,  100  cc. 

Methyl  Green. — 

Methyl  green,  i  g. 
Glacial  acetic  acid,  i  cc. 
Water,  100  cc. 

If  the  preparation  is  to  be  mounted  in  balsam,  a  slight  trace 
of  acetic  acid  and  also  a  trace  of  methyl  green  should  be  added 
to  the  absolute  alcohol  used  for  dehydrating. 

Fuchsin. — 

Fuchsin,  i  g. 

95  per  cent,  alcohol,  100  cc. 

Water,  100  cc. 

ZiehPs  Carbol  Fuchsin. — 

Fuchsin,  i  g. 

Carbolic-acid  crystals,  5  g. 

95  per  cent  alcohol,  10  cc.  1 

Water,  100  cc. 

Fuchsin  and  Iodine  Green  Mixtures. — Two  solutions  are  kept 
separate,  since  they  do  not  retain  their  efficiency  long  after  they 

are  mixed. 

0.1  g.  fuchsin  (acid). 
50  cc.  distilled  water. 


Formulce  for  Reagents  153 

j  0.1  g.  iodine  green, 
\  50  cc.  distilled  water. 

iioo  cc.  absolute  alcohol. 
I  cc.  glacial  acetic  acid. 
0.1  g.  iodine. 

Stain   in   equal   parts   of  A  and   B,     Transfer  from  the  stain 
directly  to  solution  C  and  from  C  to  xylol. 
Another  Formula. — 

Acid  fuchsin,  0.5  g. 
Water,  100  cc. 
Iodine  green,  0.5  g. 


'  Water,  100  cc. 

Mix  a  pipette  full  of  A  with  a  pipette  full  of  B ;  stain  two  to 
eight  minutes ;  dehydrate  rapidly  and  mount  in  balsam. 
Safranin. — 

Safranin,  i  g. 

95  per  cent,  alcohol,  50  cc. 

Water,  50  cc. 

Gentian  Violet. — 

Gentian  violet,  i  g. 

95  per  cent,  alcohol,  20  cc. 

Water,  80  cc. 

Anilin,  3  cc. 

Orange  G. — 

Grange  G,  i  g. 
Water,  100  cc. 

Bismark  Brown. — 

Bismark  brown,  2  g. 

70  per  cent,  alcohol,  100  cc. 

Nigrosin. — 

Nigrosin,  i  g. 
Water,  100  cc. 

Gram's  Solution. — 

Iodine,  i  g. 

Iodide  of  potassium,  2  g. 

Water,  300  cc. 


INDEX 


INDEX 


The  references  are  to  pages, 

Acid  alcohol,  formulae,  35,  43. 

^cidiomycetes,  84. 

Air  bubbles,  32, 

Alcohol,  for  fixing,  25  ;  formulae,  9, 

Algae,  63. 

Alisma,  embryo,  137. 

Alum  carmine,  40. 

Alum  cochineal,  40. 

Ammonia,  35. 

Anabaena,  6j. 

Angiosperms,  embryo,  136,  1^8;  fertiliza- 
tion, 135,  /j7,-  floral  development,  129  ; 
oogenesis,  132;  root,  139;  spermato- 
genesis, 130,  131 ;  stem,  138. 

Anilins,  formulae,  41,  152. 

Antheridia,  Chara,  72,  73;  ferns,  103, 
107;  liverworts,  gi ;  mosses,  11,  gj. 

Anthers,  fixing,  31 ;  staining,  131,  132. 

Anthoceros,  Q4. 

Anthrax,  jy. 

Apparatus,  i. 

Archegonia,  ferns,  106,  107  ;  pines,  121, 
122,  i2s;  liverworts,  g2;  mosses,  97,  98. 

Arisaema,  root  tip,  140. 

Ascomycetes,  80. 

Aspidium,  sporangia,  108. 

Aster,  18,  /j2. 

Asterella,  89,  gi. 

Azolla,  fixing,  31 ;  infiltrating,  112. 

Bacteria,  yy. 

Balsam,  mounting  in,  22. 

Basidiomycetes,  87. 

Batrachospermum,  75. 

Beggiatoa,  78. 

Bismark  brown,  45. 

Boletus,  88. 

Borax  carmine,  40. 

Botrychium,    anatomy,     109;    prothallia, 

104;   sporangia,    108. 
Bryophytes,  89. 
Bryum,  antheridia,  97  ;  capsule,  gg. 


Italic  figures  indicate  illustrations. 

Capsella,  floral  development,  129;   pod, 

132;  embryo,  138. 
Carmines,  39. 
Carnoy's  fluid,  formula,  26. 
Cataloguing  preparations,  141,  142. 
Cedar  oil,  43. 
Celloidin,  15;  method,  55. 
Centrosomes,  39. 
Chara,  paraffin  sections,  72,  73;  glycerine 

mounts,  58. 
Chlorophyceae,  65. 
Chorda,  75. 
Chromosomes,  39. 
Chromic  acid  group,  26. 
Chromo-acetic  acid,  formulae,  28. 
Cladophora,  65,  by. 
Clearing,  16,  22;  clearing  agents,  10. 
Clove  oil,  41. 

Cnicus,  floral  development,  130. 
Coelosphaerium,  63. 
Coleus,  stem  anatomy,  139. 
Collema,  88. 
Conocephalus,  89,  94. 
Coprinus,  87. 
Corn,  stem  anatomy,  139. 
Corrosive  sublimate,  30. 
Cover-glasses,  cleaning,  22. 
Crucibulum,  88. 

Cutting  paraffin,  19  ;  celloidin,  56. 
Cyanin,  formula,  8. 
Cyanin  and  erythrosin,  staining  in,  42. 
Cyanophyceae,  63. 
Cyathus,  88. 

Cypripedium,  root  tip,  140. 
Cyrtomium,  sporangia,  108. 
Cystopus,  79. 

Diatoms,  69,  yo. 
Dehydrating,  14. 
Desmids,  71,  72. 
Dichonema,  88. 


157 


58 


Methods  in  Plant  Histology 


Ectocarpus,  7j. 

Eosin,  formula,  9. 

Equisetum,  115,  116. 

Erysiphe,  83. 

Erythrosin,  formula,  8  ;  staining  in,  42. 

Eurotium,  81. 

Filicinese,  103. 

Fixative,  Mayer's  albumin,  20. 

Fixing,  general  hints,  31. 

Flemming's  fluid,  formula,  28. 

Floral  development,  129. 

Formalin,  31. 

Formulae  for  reagents,  149. 

Fuchsin  acid,  formula,  8  ;  staining  in,  44. 

Fucus,  74. 

Fuligo,  78. 

Funaria,  antheridia,  97;  capsule,  g8,  gg. 

Fungi,  77. 

Gentian  violet,  8,  43. 
Geranium,  stem  anatomy,  139. 
Glceeotrichia,  65. 
Glycerine  jelly,  58. 
Glycerine  method,  58. 
Gymnosperms,  119. 

Hsem-alum,  Mayer's,  8,  38. 

Hsematoxylin,  Boehmer's,  38 ;  Dela- 
field's,  7,  35  ;  Erlich's,  37;  Haiden- 
hain's  iron  alum,  8,  38  ;  Kleinenberg's, 
37. 

Hardening,  14. 

Hemitrichia,  y8. 

Hepaticse,  89. 

Hermann's  fluid,  formula,  29. 

Hydnum,  88. 

Hydrodictyon,  66,  67. 

Hypoxylon,  83. 

Imbedding  in  paraffin,  18;  in  celloidin, 

55. 
Iodine  green,  formula,  8  ;  staining  in,  44. 
Iris,  root  tip,  140. 
Isoetes,  117. 

Karyokinesis,  39;  in  Pinus,  119,  122,  123; 
in  Lilium,  /jj,  136;  in  Osmunda,  no; 
in  root  tips,  139. 


Killing  and  fixing,  13,  25. 

Labels,  126,  141. 

Laminaria,  75. 

Leptothrix,  78. 

Lichens,  88. 

Liliuin,  archesporium,  ijj;  fertilization, 
137 ;  fixing  anthers,  31;  fixing  ovaries, 
132;  germination  of  megaspore,  /yj, 
136;  megaspore,  134. 

List  of  preparations,  143. 

Lycoperdon,  88. 

Lycopodinese,  117. 

Lycopodium,  118. 

Marchantia,  89,  90,  91,  92,  94. 

Marsilea,  antheridium,  112;  archegonium, 

III;  imbedding  megaspores,   19,   11 2; 

procuring  material,  in. 
Merkel's  fluid,  formula,  29. 
Mica  slips,  65. 
Microscope,  i,  2. 
Microsphsera,  82. 
Microtome,  hand,  / ;  sliding,  2. 
Mildew^s,  82. 
Mosses,  97. 
Mucor,  79. 
Myxomycetes,  y8. 

Nigrosin,  45. 
Nostoc,  65. 
Nummularia,  83. 
Nuphar,  sclerotic  cells,  139. 

Oedogonium,  72. 
Orange  G,  formula,  8. 
Oscillaria,  64. 
Osmotic  apparatus,  15. 
Osmunda,    prothallia,     103;     sporangia, 
no. 

Paraffin  bath, ./,-  infiltration,  17;  removal 
of,  21;  summary  of  method,  23. 

Parmelia,  88. 

Pellia,  antheridia,  91  ;  sporophyte,  9j,- 
spores,  94. 

Peltigera,  88. 

Penicillium,  81. 

Peronospora,  81. 


Index 


159 


Peziza,  84. 

Phaeophycese,  73. 

Phycomycetes,  79. 

Physcia,  88. 

Picric  acid,  14,  30. 

Pinus,  embryo,  121 ;  leaves,  124  ;  oogen- 
esis, 121;  spermatogenesis,  119,  120; 
stems  and  roots,  124,  125;  ventral  ca- 
nal cell,  123. 

Podophyllum,  root  tip,  140. 

Pollen,  of  Lilium,  131 ;  of  Pinus,  120. 

Polyporus,  88. 

Polysiphonia,  jb. 

Polytrichum,  antheridia,  97. 

Prothallia,  11,  26,  103,  /oj,  ///,  112. 

Protonema,  li,  loi. 

Pteridophytes,  103. 

Pteris,  antheridia,  107;  archegonia,  106; 
prothallia,  103,  lo^;  rhizome,  no; 
sporangia,  108. 

Ptilidium,  qo. 

Puccinia,  84,  83. 

Pythium,  81. 

Ranunculus,  138. 

Razor,  4. 

Reagents,  7. 

Rhodophycese,  75. 

Riccia,  89,  90J 

Ricciocarpus,  89,  9j. 

Rivularia,  64. 

Roots,  of  seed  plants,  138,  I3g;  of  Equi- 

setum,  116;  tips  for  karyokinesis,  139, 

140. 
Rumex,  138. 

Saccharomyces,  80. 

Safranin,  formulae,  8,  42;  safranin-gentian 
violet-orange  method,  42. 


Sagittaria,  embryo 
Salix,  130,  131. 
Saprolegnia,  81. 
Schizomycetes,  77. 
Scytonema,  65. 
Selaginella,  113,  7/7. 
Silphium,  ovules,  1 19 
Spermatophytes,  119. 


37- 


Sphagnum,  100. 

Spirillum,  "jy. 

Spirogyra,  13;  fixing,  26,  67,  68;  imbed- 
ding, 69. 

Sporangia,  of  ferns,  108. 

Staining,  33  ;  analytical  value,  49 ;  Fi- 
scher's experiments,  50;  general  re- 
mark, 47;  practical  hints,  53;  Stras- 
burger's  theory,  48. 

Stains,  formulae,  7. 

Staphylococcus,  77. 

Starch,  11. 

Stemonitis,  78. 

Stem,  of  seed  plants,  138. 

Stender  dish,  5. 

Stereum,  88. 

Sticta,  88. 

Taraxacum,  129. 
Teleutospores,  83,  86. 
Temporary  mounts,  11. 
Thamnidium,  81. 
Tolypothrix,  b^. 
Tradescantia,  root  tips,  140 
Tremella,  88. 
Trichia,  78. 
Turn  table,  5. 

Uncinula,  82, 
Uredospores,  83,  86. 
Ustilago,  86. 
Ustilina,  83. 
Usnea,  88. 

Vaucheria,  27,  bb. 
Volvox,  65. 

Wasserbliithe,  6j. 

Washing,  14. 

Webbera,  archegonia,  q7. 

Xanthium,  stem  anatomy,  139. 

Xylaria,  83. 

Xylol,  removal  of,  21. 

Zea,  stem  anatomy,  139. 
Zygnema,  bg. 


UC  SOUTHERN  REGIONAL  UBRARY  FAaUTY 

iillllllil 

A    000  865  422    0 


STAi£,^...aAL  SCHOOL, 


